High-density ion transport measurement biochip devices and methods

ABSTRACT

The present invention includes biochips for the measurement of cellular ion channels and methods of use and manufacture. The biochips of the present invention have enhanced sealing capabilities provided in part by chemically modifying the surface of the biochip surface or substrate or by exposure to an ionized gas. The present invention also includes novel cartridges for biochips.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent applicationSer. No. 11/033,015 filed on Jan. 10, 2005, which is acontinuation-in-part of U.S. patent application Ser. No. 10/858,339filed on Jun. 1, 2004, which claims benefit of priority to U.S. PatentApplication Ser. No. 60/474,508 filed on May 31, 2003; U.S. patentapplication Ser. No. 11/033,015 is also a continuation-in-part of U.S.patent application Ser. No. 10/760,886 filed on Jan. 20, 2004, which isa continuation-in-part of U.S. patent application Ser. No. 10/428,565filed on May 2, 2003, which claims benefit of priority to U.S. PatentApplication Ser. No. 60/380,007 filed on May 4, 2002; U.S. patentapplication Ser. No. 11/033,015 is also a continuation-in-part of U.S.patent application Ser. No. 10/642,014 filed on Aug. 16, 2003, which isa continuation-in-part of U.S. patent application Ser. No. 10/351,019filed on Jan. 23, 2003, which claims benefit of priority of U.S. PatentApplication Ser. No. 60/351,849 filed on Jan. 24, 2002; U.S. patentapplication Ser. No. 11/033,015 is also a continuation-in-part of U.S.patent application Ser. No. 10/104,300 filed on Mar. 22, 2002, whichclaims benefit of priority to U.S. Patent Application Ser. No.60/311,327 filed on Aug. 10, 2001 and claims benefit of priority to U.S.Patent Application Ser. No. 60/278,308 filed on Mar. 24, 2001; U.S.patent application Ser. No. 11/033,015 also claims benefit of priorityof U.S. Patent Application Ser. No. 60/535,461 filed on Jan. 10, 2004and claims benefit of priority of U.S. Patent Application Ser. No.60/585,822 filed on Jul. 6, 2004; this application is also acontinuation-in-part of U.S. patent application Ser. No. 11/153,825filed on Jun. 15, 2005; the present application incorporates byreference herein each of the above referenced U.S. patent applications.

TECHNICAL FIELD

The present invention relates generally to the field of ion transportdetection (“patch clamp”) systems and methods, more particularly thepresent invention includes a novel ion channel chip including a highresistance seal for use with automated or high throughput systems andmethods.

BACKGROUND

Ion transports are channels, transporters, pore forming proteins, orother entities that are located within cellular membranes and regulatethe flow of ions across the membrane. Ion transports participate indiverse processes, such as generating and timing of action potentials,synaptic transmission, secretion of hormones, contraction of musclesetc. Ion transports are popular candidates for drug discovery, and manyknown drugs exert their effects via modulation of ion transportfunctions or properties. For example, antiepileptic compounds such asphenytoin and lamotrigine which block voltage dependent sodium iontransports in the brain, anti-hypertension drugs such as nifedipine anddiltiazem which block voltage dependent calcium ion transports in smoothmuscle cells, and stimulators of insulin release such as glibenclamideand tolbutamine which block an ATP regulated potassium ion transport inthe pancreas.

One popular method of measuring an ion transport function or property isthe patch-clamp method, which was first reported by Neher, Sakmann andSteinback (Pflueger Arch. 375:219-278 (1978)). This first report of thepatch clamp method relied on pressing a glass pipette containingacetylcholine (Ach) against the surface of a muscle cell membrane, wherediscrete jumps in electrical current were attributable to the openingand closing of Ach-activated ion transports.

The method was refined by fire polishing the glass pipettes and applyinggentle suction to the interior of the pipette when contact was made withthe surface of the cell. Seals of very high resistance (between about 1and about 100 giga ohms) could be obtained. This advancement allowed thepatch clamp method to be suitable over voltage ranges which iontransport studies can routinely be made.

A variety of patch clamp methods have been developed, such as wholecell, vesicle, outside-out and inside-out patches (Liem et al.,Neurosurgery 36:382-392 (1995)). Additional methods include whole cellpatch clamp recordings, pressure patch clamp methods, cell free iontransport recording, perfusion patch pipettes, concentration patch clampmethods, perforated patch clamp methods, loose patch voltage clampmethods, patch clamp recording and patch clamp methods in tissue samplessuch as muscle or brain (Boulton et al, Patch-Clamp Applications andProtocols, Neuromethods V. 26 (1995), Humana Press, New Jersey).

These and later methods relied upon interrogating one sample at a timeusing large laboratory apparatus that require a high degree of operatorskill and time. Attempts have been made to automate patch clamp methods,but these have met with little success. Alternatives to patch clampmethods have been developed using fluorescent probes, such ascumarin-lipids (cu-lipids) and oxonol fluorescent dyes (Tsien et al.,U.S. Pat. No. 6,107,066, issued August 2000). These methods rely uponchange in polarity of membranes and the resulting motion of oxonolmolecules across the membrane. This motion allows for the detection ofchanges in fluorescence resonance energy transfer (FRET) betweencu-lipids and oxonol molecules. Unfortunately, these methods do notmeasure ion transport directly but measure the change of indirectparameters as a result of ionic flux. For example, the characteristicsof the lipid used in the cu-lipid can alter the biological and physicalcharacteristics of the membrane, such as fluidity and polarizability.

Thus, what is needed is a simple device and method to measure iontransport directly. Preferably, these devices would utilize patch clampdetection methods because these types of methods represent a goldstandard in this field of study. The present invention provides thesedevices and methods particularly miniaturized devices and automatedmethods for the screening of chemicals or other moieties for theirability to modulate ion transport functions or properties.

BRIEF SUMMARY OF THE INVENTION

The present invention recognizes that the determination of one or moreion transport functions or properties using direct detection methods,such as patch-clamp, whole cell recording, or single channel recording,are preferable to methods that utilize indirect detection methods, suchas fluorescence-based detection systems.

The present invention provides biochips for ion transport measurement,ion transport measuring devices that comprise biochips, and methods ofusing the devices and biochips that allow for the direct analysis of iontransport functions or properties. The present invention providesbiochips, devices, apparatuses, and methods that allow for automateddetection of ion transport functions or properties. The presentinvention also provides methods of making biochips and devices for iontransport measurement that reduce the cost and increase the efficiencyof manufacture, as well as improve the performance of the biochips anddevices. These biochips and devices are particularly appropriate forautomating the detection of ion transport functions or properties,particularly for screening purposes.

A first aspect of the present invention is a biochip device for iontransport measurement. A biochip device comprises an upper chamber piecethat comprises one or more upper chambers, and a biochip that comprisesat least one ion transport measuring means. In one preferred embodimentof this aspect of the present invention, a biochip device also comprisesat least one conduit that that can be positioned to engage the one ormore upper chambers, where the conduit comprises an electrode or canprovide an electrolyte bridge to an electrode.

A second aspect of the present invention is a biochip device having oneor more flow-through lower chambers. The device comprises an upperchamber piece that comprises one or more upper chambers, a biochip thatcomprises at least one ion transport measuring means, and at least onelower chamber base piece that comprises at least two conduits thatconnect with at least one lower chamber.

A third aspect of the invention is biochip devices that are adapted formicroscope stages. The devices comprise an upper chamber piece thatcomprises one or more upper chambers, a biochip that comprises at leastone ion transport measuring means, and at least one lower chamber basepiece, in which the bottom surface of the lower chamber base piece istransparent. Preferably, the device also includes a base-plate adaptedto a microscope stage into which a lower chamber base piece can fit.

A fourth aspect of the invention is methods of making an upper chamberpiece for a biochip device for ion transport measurement. In onepreferred embodiment of this aspect of the present invention, an upperchamber piece can be molded as two pieces, an upper well portion pieceand a well hole portion piece. Preferably, a well hole portion piececomprises at least one groove into which at least one electrode can beinserted. After insertion of the electrode, the upper well portion pieceand the well hole portion piece are attached to form an upper chamberpiece. In another embodiment of this aspect, an upper chamber piece canbe molded as a single piece, where an electrode, such as a wireelectrode, can be positioned in a mold and then the upper chamber piececan be molded around it. In yet another preferred embodiment of thisaspect, an upper chamber piece can be molded as a single piece withoutan electrode.

A fifth aspect of the invention is methods for making chips comprisingion transport measuring holes. An ion transport measuring hole can befabricated by laser drilling one or more counterbores, and then laserdrilling a through hole through the one or more counterbores.

A sixth aspect of the invention is ion transport measuring devices thatcomprise an inverted chip comprising ion transport measuring holes. Achip used in inverted orientation can comprise one or more ion transportmeasuring holes that are fabricated by laser drilling of one or morecounterbores and a through hole through the one or more counterbores.

A seventh aspect of the invention is methods of treating ion transportmeasuring chips to enhance their sealing properties. In one aspect ofthe present invention, the chip or substrate comprising an ion transportmeasuring means is modified to become more electronegative and/orsmoother. In another aspect of the present invention, the chip orsubstrate comprising the ion transport measuring means is modifiedchemically, such as with acids, bases, or a combination thereof.Treatment of chips of the present invention with chemical solution canbe performed using treatment racks that fit into vessels that hold thechemical solutions and can hold multiple glass chips while allowingaccess of the chemical solutions to the chip surfaces.

An eighth aspect of the invention is a method to measure surface energyon a surface, such as the surface of a chemically-treated ion transportmeasurement biochip. The surface energy measurement can be used toevaluate the hydrophilicity of a biochip biochip of the presentinvention that has been chemically treated to improve its electricalsealing properties, such as, for example, at chip that has been treatedwith base. It can also be used for any surface characterization purposewhere a measurement of surface energy or hydrophilicity is desired.

A ninth aspect of the invention is the substrates, biochips, devices,apparatuses, and/or cartridges comprising ion transport measuring meanswith enhanced electric seal properties. In preferred embodiments, atleast a portion of at least one chip that comprises at least one iontransport measuring means has been treated with at least one base, atleast one acid, or both.

A tenth aspect of the present invention is a method for storing thesubstrates, biochips, cartridges, apparatuses, and/or devices comprisingion transport measuring means with enhanced electrical seal properties.

An eleventh aspect of the present invention is a method for shipping thesubstrates, biochips, cartridges, apparatuses, and/or devices comprisingion transport measuring means with enhanced electrical seal properties.

A twelfth aspect of the invention is methods for assembling devices andcartridges of the present invention. The methods include attaching anupper chamber piece to a biochip that comprises at least one iontransport measuring means using a UV adhesive. Preferably, the chip hasbeen chemically treated to enhance its electrical sealing properties.During UV activation of the adhesive, at least a portion of the biochipis masked to prevent UV irradiation of ion transport measuring means onthe chip.

A thirteenth aspect of the present invention is a method of producingbiochips comprising ion transport measuring means by fabricating thebiochips as detachable units of a large sheet. Ion transport measuringholes can be made by wet etching and laser drilling appropriatesubstrates, and the sheet can be scored with a laser such that portionsof the sheet having a desired number of ion transport measuring holescan be separated along the score lines. In some embodiments, upperchamber pieces are attached to the substrate sheet after the fabricationof holes and before separation of sections of the sheet. In this case,the detachable units that are separated to produce devices comprisecartridges having upper chambers attached to an ion transport measuringchip.

A fourteenth aspect of the invention is a method of producing highdensity ion transport measuring chips. The ion transport measuring chipspreferably have more than 16 ion transport measuring holes, and wellscan be fabricated in a chip using wet etching, followed by laserdrilling of ion transport measuring holes through the bottoms of thewells.

A fifteenth aspect of the invention is a biochip device for iontransport measurement comprising fluidic channel upper and lowerchambers. The fluidic channels have apertures that are aligned with iontransport measuring holes on the chip. The fluidic channels can beconnected to sources for generating or promoting fluid flow, such aspumps, pressure sources, and valves. The fluidic channels preferablyprovide electrolyte bridge to one or more electrodes that can be used inion transport measurement.

A sixteenth aspect of the present invention is methods of preparingcells for ion transport measurement. The methods include the use offilters that can allow the passage of single cells through their poresand monitoring of cell health parameters important forelectrophysiological measurements.

A seventeenth aspect of the present invention is a logic and programthat uses a pressure profile to direct an ion transport measurementapparatus to achieve and maintain a high-resistance electrical seal. Thelogic can follow decision pathways based on information from electricalmeasurements made by ion transport measuring electrodes in a feedbacksystem.

An eighteenth aspect of the present invention is an ion channel chiphaving a plastic substrate or surface that is chemically modified andmethods of use. Chemical modification may provide enhanced sealingproperties. Modification of the plastic substrate or surface may occurby treatment with an ionized gas, by interaction with laser or otherhigh energy radiation, by chemical reaction, or by any combination ofthese.

A nineteenth aspect of the present invention is an ion channel chip orbiochip utilizing a lipid layer or lipid bilayer for enhancing thesealing properties. The lipid bilayer is capable of interacting with themembrane of a cell having to facilitate measurement of a cell's ionchannel.

A twentieth aspect of the present invention is a method of making apre-assembled ion transport measurement cartridge and method of use. Achip without a hole or aperture may be provided in the cartridge. Thecartridge may be oriented such that a drill may form an aperture throughthe chip. The cartridge may then be treated for surface modification topromote the formation of tight seals for ion transport measurement.

A twenty-first aspect of the present invention is a method of layering aplastic chip with a thin sheet of glass and method of use. Holes orapertures may be drilled through the layered chip using a laser drill orwet etching.

A twenty-second aspect of the present invention is a method ofprotecting fragile devices and parts by packing and shipping the devicesor parts in a fluid such as water.

A twenty-third aspect of the present invention is a method of laser beamsplitting and method of use to laser drill holes in ion channel chipsand methods of use. The method may utilize a homogenizer to obtain a“top hat” power profile from a laser beam source. The laser beam mayoptionally be masked to provide additional profiles. The laser beam maythen be split to form two or more laser beamlets.

A twenty-fourth aspect of the present invention is a method of makingglass more readily wet etched, ion channel chips made using this methodand methods of use thereof. The method may include exposing the glass toa laser beam in the ultra-violet (UV) range.

A twenty-fifth aspect of the present invention are methods for bondingglass to glass and products produced by these methods. The methods mayinclude treating one or more surfaces of the glass with an acid or base,contacting the glass surfaces together and heating the glass.Alternatively, NaSiO4 may be applied between the glass surfaces andheated.

A twenty-sixth aspect of the present invention is an in situ method ofmaking a cartridge for use in ion transport measurement and methods ofuse. The cartridge may include a chip with gaskets defining top andbottom chamber perimeters.

A twenty-seventh aspect of the present invention is a gasket for use iontransport measurement and methods of use thereof. The gasket may engagethe chip and define a boundary of a chamber.

A twenty-eighth aspect of the present invention is a system forautomated processing of chips for use in ion transport measurement. Themethod may include exposing the chip to a treatment solution andexposing the chip to a negative or positive pressure.

A twenty-ninth aspect of the present invention is a method for making asilicon-based chip with laser-drilled holes and surface modifications.The method may include providing a silicon based chip, drilling holes inthe chip and treating the chip to form surface modifications.

A thirtieth aspect of the present invention is a device that can holdchips for methods of treating chips and/or for storage of chips. Thedevice may include a bottom structure having chip holding structures,and a hinged top structure.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts views an upper chamber piece of the present inventioncomprising glue spillage grooves and alignment bump.

FIG. 2 depicts a biochip device of the present invention that is adaptedto a microscope stage. (A) Top view and (B) bottom view of a base platecut from aluminum stock. The holes are threaded except for the fourholes closest to the corners of the square-cut carve-out. The fourunthreaded holes are sized to accept a press-in 1 mm socket connector.

FIG. 3 is a cross-sectional view of a device of the present inventionhaving a flow through lower chamber.

FIG. 4 provides drawings of a design of a flow through chamber lowerbase piece of a device of the present invention.

FIG. 5 depicts a biochip device of the present invention having flowthrough lower chambers that is adapted to fit a microscope stage. A)view of a plastic lower chamber piece from behind the electrodes, B) azoomed-in side view of the lower chamber piece to demonstrate cuts inthe top side, inflow and outflow tubes, and an edge for gasketalignment, C) the lower chamber piece installed in a base late.

FIG. 6 A) depicts the lower chamber piece of FIG. 5 viewed from behindthe electrode array with connectors on top (brass inserts) and wiresextending into the fluidics of each chamber below. B) A schematicdemonstrates shows the 16× device fully assembled with a cartridgewithin.

FIG. 7 shows glass tubing inserted into the plastic lower chamber pieceshown in FIG. 6. The tubing has been cemented into place with 20 minuteepoxy glue. Silicone tubing is ten pressed over the glass tubing tocomplete the conduit to deliver fluids into the lower chambers.

FIG. 8 depicts a PDMS gasket viewed from the top (A), from the side (B)and (C) a schematic demonstrating the formation of the inner wells bythe holes within the gasket.

FIG. 9 shows a cartridge of the present invention (black item) is shownin relation to the rest of the parts of a device adapted for amicroscope (A) and after installation into the device (B).

FIG. 10 (A) shows a clamp part upside down to illustrate the cutout thatfits the cartridge. The top view of the clamp on the cartridge (B)reveals the presence of an array of top chamber electrodes that reachinto the cartridge wells (C).

FIG. 11 depicts an upper chamber piece of the present invention that ismade from an upper well portion piece and a well hole portion piece. (A)is the upper well portion piece is shown above the well hole portionpiece. (B) is the upper well portion piece is shown fitted on the wellhole portion piece, with the groove visible along the back of the wells.

FIG. 12 is a graph that illustrates that a decreasing hole depth andwidening the exit hole decreases Ra.

FIG. 13 is a graph illustrating that thinner chips (“K-configurationchips”) have a lower Re than those with greater hole depth.

FIG. 14 depicts treatment fixtures for chemically treating chips anddevices. (A) shows a single layer treatment fixture that can fit into aglass jar containing acid, base, or other chemical solutions. (B) showsthe stacked fixture.

FIG. 15 shows a shipping fixture for cartridges of the presentinvention.

FIG. 16 shows a shipping fixture for chips of the present invention.

FIG. 17 depicts a glass chip with multiple ion transport units (below)that can be attached to a multichamber upper chamber piece. Cartridgeswith a smaller number of units (above) can be separated from the largermulticartridge unit.

FIG. 18 depicts a high density array chip of the present invention.

FIG. 19 depicts a cross sectional view of a high density array chip ofthe present invention.

FIG. 35 depicts one embodiment of an ion transport measuring chip madefrom an MCP. A) Top view. B) cross-sectional view showing etchedmicrowells and through-holes.

FIG. 36 depicts a cross-sectional view of a portion of a chip having ahydrophobic coating and microwells.

FIG. 37 depicts two embodiments of a flexible chip of the presentinvention. A) the chip extends between two spools, with the assay arealocalized to the extended portion of the chip between them. B) the assayarea of the chip corresponds to a portion chip that curves over a spool,which can comprise or engage lower chambers for ion transport assays.

FIG. 38 depicts preferred embodiments of the present invention: ionchannel measuring devices that comprise theta tubing. A) a segment oftheta tubing shown “face on” in which the opening for laser access (usedin making the hole) is shown. B) an ion transport measuring devicecomprising multiple theta units arranged vertically. The upper and lowerchambers of each unit have separate conduit attachments for ES and IS,respectively. “U” labels conduits leading to and away from upperchambers. “L” labels conduits leading to and away from lower chambers.Pressure can be applied from either the inflow or outflow lower chamberconduits. C) an ion transport measuring device comprising multiple thetaunits arranged side-by-side. Although conduits connecting with only oneof the units are shown, each of the upper and lower chambers of eachunit have separate conduit attachments for ES and IS, respectively.

FIG. 39 is a top view of one embodiment of the present inventioncomprising a chip having a fluidic channel on its upper surface that hasopenings localized to ion transport measuring holes.

FIG. 40 is a cross-sectional depiction of one embodiment of an iontransport measuring device comprising a fluidic pipe compound deliverysystem.

FIG. 41 depicts one embodiment of an ion transport measuring devicehaving a nozzle compound delivery system.

FIG. 42 depicts one embodiment of an ion transport measuring devicehaving a compound delivery plate that delivers compounds to cells sealedat ion transport measuring hole on the lower side of a chip.

FIG. 43 depicts one embodiment of an ion transport measuring device inwhich compound is delivered by fluid dispensing tips at ion transportmeasuring sites. In this embodiment, an electrode traverses the surfaceof the chip. A hydrophobic layer coats the electrode, except in theimmediate vicinity of microwells. A) cells have been added to an upperchamber channel comprising ES. B) cells seal to ion transport measuringholes within microwells that access the channel. C) compound drops aredispensed directly over the ion transport measuring sites. D) compoundsolution floods the microwell, but does not flow into neighboringmicrowells.

FIG. 44 depicts one embodiment of an ion transport measuring devicehaving a single flow-through upper chamber.

FIG. 45 depicts one embodiment of an ion transport measuring devicehaving a single flow-through upper chamber and a single flow-throughlower chamber.

FIG. 46 depicts one embodiment of an ion transport measuring devicehaving a single flow-through upper chamber and a multiple lowerchambers. A) cross sectional view. B) top view.

FIG. 47 depicts one embodiment of a single use chip of the presentinvention. A) top view. B) cross sectional view.

FIG. 48 depicts one embodiment of a chip of the present invention inwhich wax forms the upper chambers. A) top view. B) cross sectionalview.

FIG. 49 depicts one embodiment of a chip of the present invention inwhich O-rings form the upper chambers. A) top view. B) cross sectionalview.

FIG. 50 depicts a schematic of a (A) 16×24 hole array at a pitch of 4.5mm for an ion channel chip design and (B) a 16×1 hole array at a pitchof 4.5 mm for an ion channel chip design.

FIG. 51 depicts the production of 4 laser drilled holes at one time tomake a plurality of holes for ion channel chip designs, such as in FIG.50(A).

FIG. 52 depicts the production of 9 laser drilled holes at one time tomake a plurality of holes for ion channel chip designs, such as in FIG.50(A).

FIG. 53 depicts one aspect of the present invention wherein a chipengaged with gaskets to form upper chambers and lower chambers.

FIG. 54 depicts one aspect of the present invention wherein a gasket isfor use in ion transport measurement. (A) represents a top view of oneaspect of the present invention wherein the gasket includes o-ringstructures and holes for use in engaging a chip for use in ion transportmeasurement and for providing negative pressure to secure the chip tothe gasket to assist in preventing or reducing cross-talk betweenrecording sites during ion transport measurement. Vents can be providedto assist in modulating the negative pressure used to secure the chip tothe gasket and remove fluids from the gasket. (B) depicts a partialcross section along A-A.

FIG. 55 depicts one aspect of the present invention wherein a system,device and method of processing, handling and assembling chips for usein ion transport measurement are provided.

FIG. 56 depicts on aspect of the present invention wherein a device forholding chips for storage and/or treatment are provided.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Generally, the nomenclatureused herein and the manufacture or laboratory procedures described beloware well known and commonly employed in the art. Conventional methodsare used for these procedures, such as those provided in the art andvarious general references. Terms of orientation such as “up” and “down”, “top” and “bottom”, “upper” or “lower” and the like refer toorientation of parts during use of a device. Where a term is provided inthe singular, the inventors also contemplate the plural of that term.Where there are discrepancies in terms and definitions used inreferences that are incorporated by reference, the terms used in thisapplication shall have the definitions given herein. As employedthroughout the disclosure, the following terms, unless otherwiseindicated, shall be understood to have the following meanings:

“Ion transport measurement” is the process of detecting and measuringthe movement of charge and/or conducting ions across a membrane (such asa biological membrane), or from the inside to the outside of a particleor vice versa. In most applications, particles will be cells,organelles, vesicles, biological membrane fragments, artificialmembranes, bilayers or micelles. In general, ion transport measurementinvolves achieving a high resistance electrical seal of a membrane orparticle with a surface that has an aperture, and positioning electrodeson either side of the membrane or particle to measure the current and/orvoltage across the portion of the membrane sealed over the aperture, or“clamping” voltage across the membrane and measuring current applied toan electrode to maintain that voltage. However, ion transportmeasurement does not require that a particle or membrane be sealed to anaperture if other means can provide electrode contact on both sides of amembrane. For example, a particle can be impaled with a needle electrodeand a second electrode can be provided in contact with the solutionoutside the particle to complete a circuit for ion transportmeasurement. Several techniques collectively known as “patch clamping”can be included as “ion transport measurement”.

An “ion transport measuring means” refers to a structure that can beused to measure at least one ion transport function, property, or achange in ion channel function, property in response to variouschemical, biochemical or electrical stimuli. Typically, an ion transportmeasuring means is a structure with an opening that a particle can sealagainst, but this need not be the case. For example, needles as well asholes, apertures, capillaries, and other detection structures of thepresent invention can be used as ion transport measuring means. An iontransport measuring means is preferably positioned on or within abiochip or a chamber. Where an ion transport measuring means refers to ahole or aperture, the use of the terms “ion transport measuring means”“hole” or “aperture” are also meant to encompass the perimeter of thehole or aperture that is in fact a part of the chip or substrate (orcoating) surface (or surface of another structure, for example, achannel) and can also include the surfaces that surround the interiorspace of the hole that is also the chip or substrate (or coating)material or material of another structure that comprises the hole oraperture.

A “hole” is an aperture that extends through a chip. Descriptions ofholes found herein are also meant to encompass the perimeter of the holethat is in fact a part of the chip or substrate (or coating) surface,and can also include the surfaces that surround the interior space ofthe hole that is also the chip or substrate (or coating) material. Thus,in the present invention, where particles are described as beingpositioned on, at, near, against, or in a hole, or adhering or fixed toa hole, it is intended to mean that a particle contacts the entireperimeter of a hole, such that at least a portion of the surface of theparticle lies across the opening of the hole, or in some cases, descendsto some degree into the opening of the whole, contacting the surfacesthat surround the interior space of the hole.

A “patch clamp detection structure” refers to a structure that is on orwithin a biochip or a chamber that is capable of measuring at least oneion transport function or property via patch clamp methods.

A “chip” is a solid substrate on which one or more processes such asphysical, chemical, biochemical, biological or biophysical processes canbe carried out. Such processes can be assays, including biochemical,cellular, and chemical assays; ion transport or ion channel function oractivity determinations, separations, including separations mediated byelectrical, magnetic, physical, and chemical (including biochemical)forces or interactions; chemical reactions, enzymatic reactions, andbinding interactions, including captures. The micro structures ormicro-scale structures such as, channels and wells, electrode elements,electromagnetic elements, may be incorporated into or fabricated on thesubstrate for facilitating physical, biophysical, biological,biochemical, chemical reactions or processes on the chip. The chip maybe thin in one dimension and may have various shapes in otherdimensions, for example, a rectangle, a circle, an ellipse, or otherirregular shapes. The size of the major surface of chips of the presentinvention can vary considerably, for example, from about 1 mm² to about0.25 m². Preferably, the size of the chips is from about 4 mm² to about25 cm² with a characteristic dimension from about 1 mm to about 5 cm.The chip surfaces may be flat, or not flat. The chips with non-flatsurfaces may include wells fabricated on the surfaces.

A “biochip” is a chip that is useful for a biochemical, biological orbiophysical process. In this regard, a biochip is preferablybiocompatible.

A “chamber” is a structure that comprises or engages a chip and that iscapable of containing a fluid sample. The chamber may have variousdimensions and its volume may vary between 0.001 microliters and 50milliliters. In devices of the present invention, an “upper chamber” isa chamber that is above a biochip, such as a biochip that comprises oneor more ion transport measuring means. In the devices of the presentinvention, a chip that comprises one or more ion transport measuringmeans can separate one or more upper chambers from one or more lowerchambers. During use of a device, an upper chamber can contain measuringsolutions and particles or membranes. An upper chamber can optionallycomprise one or more electrodes. In devices of the present invention, a“lower chamber” is a chamber that is below a biochip. During use of adevice, a lower chamber can contain measuring solutions and particles ormembranes. A lower chamber can optionally comprise one or moreelectrodes.

A “lower chamber piece” is a part of a device for ion transportmeasurement that forms at least a portion of one or more lower chambersof the device. A lower chamber portion piece preferably comprises atleast a portion of one or more walls of one or more lower chambers, andcan optionally comprise at least a portion of a bottom surface of one ormore lower chambers, and can optionally comprise one or more conduitsthat lead to one or more lower chambers, or one or more electrodes.

A “lower chamber base piece” is a part of a device for ion transportmeasurement that forms the bottom surface of one or more lower chambersof the device. A lower chamber base piece can also optionally compriseone or more walls of one or more lower chambers, one or more conduitsthat lead to one or more lower chambers, or one or more electrodes.

As used herein, a “platform” is a surface on which a device of thepresent invention can be positioned. A platform can comprises the bottomsurface of one or more lower chambers of a device.

An “upper chamber piece” is a part of a device for ion transportmeasurement that forms at least a portion of one or more upper chambersof the device. An upper chamber piece can comprise one or more walls ofone or more upper chambers, and can optionally comprise one or moreconduits that lead to an upper chamber, and one or more electrodes.

An “upper chamber portion piece” is a part of a device for ion transportmeasurement that forms a portion of one or more upper chambers of thedevice. An upper chamber portion piece can comprise at least a portionof one or more walls of one or more upper chambers, and can optionallycomprise one or more conduits that lead to an upper chamber, or one ormore electrodes.

A “well” is a depression in a substrate or other structure. For example,in devices of the present invention, upper chambers can be wells formedin an upper chamber piece. The upper opening of a well can be of anyshape and can be of an irregular conformation. The walls of a well canextend upward from the lower surface of a well at any angle or in anyway. The walls can be of any shape and can be of an irregularconformation, that is, they may extend upward in a sigmoidal orotherwise curved or multi-angled fashion.

A “well hole” is a hole in the bottom of a well. A well hole can be awell-within-a well, having its own well shape with an opening at thebottom.

A “well hole piece” is a part of a device for ion transport measurementthat comprises one or more well holes of the wells of the device.

When wells or chambers (including fluidic channel chambers) are “inregister with” ion transport measuring means of a chip, there is aone-to-one correspondence of each of the referenced wells or chambers toeach of the referenced ion transport measuring means, and an iontransport measuring means is positioned so that it is exposed to theinterior of the well or chamber it is in register with, such that iontransport measurement can be performed using the chamber as acompartment for measuring current or voltage through or across the iontransport measuring means.

A “port” is an opening in a wall or housing of a chamber through which afluid sample or solution can enter or exit the chamber. A port can be ofany dimensions, but preferably is of a shape and size that allows asample or solution to be dispensed into a chamber by means of a pipette,syringe, or conduit, or other means of dispensing a sample.

A “conduit” is a means for fluid to be transported from one area toanother area of a device, apparatus, or system of the present inventionor to another structure, such as a dispensation or detection device. Insome aspects, a conduit can engage a port in the housing or wall of achamber. In some aspects, a part of a device, such as, for example, anupper chamber piece or a lower chamber piece can comprise conduits inthe form of tunnels that pass through the upper chamber piece andconnect, for example, one area or compartment with another area orcompartment. A conduit can be drilled or molded into a chip, chamber,housing, or chamber piece, or a conduit can comprise any material thatpermits the passage of a fluid through it, and can be attached to anypart of a device. In one preferred aspect of the present invention, aconduit extends through at least a portion of a device, such as a wallof a chamber, or an upper chamber piece or lower chamber piece, andconnects the interior space of a chamber with the outside of a chamber,where it can optionally connect to another conduit, such as tubing. Somepreferred conduits can be tubing, such as, for example, rubber, Teflon,or Tygon tubing. A conduit can be of any dimensions, but preferablyranges from 10 microns to 5 millimeters in internal diameter.

A “device for ion transport measurement” or an “ion transport measuringdevice” is a device that comprises at least one chip that comprises oneor more ion transport measuring means, at least a portion of at leastone upper chamber, and, preferably, at least a portion of at least onelower chamber. A device for ion transport measurement preferablycomprises one or more electrodes, and can optionally comprise conduits,particle positioning means, or application-specific integrated circuits(ASICs).

A “cartridge for ion transport measurement” comprises an upper chamberpiece and at least one biochip comprising one or more ion transportmeasuring means attached to the upper chamber piece, such that the oneor more ion transport measuring means are in register with the upperchambers of the upper chamber piece.

An “ion transport measuring unit” is a portion of a device thatcomprises at least a portion of a chip having an ion transport measuringmeans and an upper chamber, where the ion transport measuring meansconnects the upper chamber with a portion of a lower chamber.

A “measuring solution” is an aqueous solution containing electrolytes,with pH, osmolarity, and other physical-chemical traits that arecompatible with conducting function of the ion transports to bemeasured.

An “intracellular solution” is a measuring solution used in the upper orlower chamber that is compatible with the electrolyte composition andphysical-chemical traits of the intracellular content of a living cell.

An “extracellular solution” is a measuring solution used in the upper orlower chamber that is compatible with the electrolyte composition andphysical-chemical traits of the extracellular content of a living cell.

To be “in electrical contact with” means one component is able toreceive and conduct electrical signals (voltage, current, or change ofvoltage or current) from another component.

An “ion transport” can be any protein or non-protein moiety thatmodulates, regulates or allows transfer of ions across a membrane, suchas a biological membrane or an artificial membrane. Ion transportinclude but are not limited to ion channels, proteins allowing transportof ions by active transport, proteins allowing transport of ions bypassive transport, toxins such as from insects, viral proteins or thelike. Viral proteins, such as the M2 protein of influenza virus can forman ion channel on cell surfaces.

A “particle” refers to an organic or inorganic particulate that issuspendable in a solution and can be manipulated by a particlepositioning means. A particle can include a cell, such as a prokaryoticor eukaryotic cell, or can be a cell fragment, such as a vesicle or amicrosome that can be made using methods known in the art. A particlecan also include artificial membrane preparations that can be made usingmethods known in the art. Preferred artificial membrane preparations arelipid bilayers, but that need not be the case. A particle in the presentinvention can also be a lipid film, such as a black-lipid film (see,Houslay and Stanley, Dynamics of Biological Membranes, Influence onSynthesis, Structure and Function, John Wiley & Sons, New York (1982)).In the case of a lipid film, a lipid film can be provided over a hole,such as a hole or capillary of the present invention using methods knownin the art (see, Houslay and Stanley, Dynamics of Biological Membranes,Influence on Synthesis, Structure and Function, John Wiley & Sons, NewYork (1982)). A particle preferably includes or is suspected ofincluding at least one ion transport or an ion transport of interest.Particles that do not include an ion transport or an ion transport ofinterest can be made to include such ion transport using methods knownin the art, such as by fusion of particles or insertion of iontransports into such particles such as by detergents, detergent removal,detergent dilution, sonication or detergent catalyzed incorporation(see, Houslay and Stanley, Dynamics of Biological Membranes, Influenceon Synthesis, Structure and Function, John Wiley & Sons, New York(1982)). A microparticle, such as a bead, such as a latex bead ormagnetic bead, can be attached to a particle, such that the particle canbe manipulated by a particle positioning means.

A “cell” refers to a viable or non-viable prokaryotic or eukaryoticcell. A eukaryotic cell can be any eukaryotic cell from any source, suchas obtained from a subject, human or non-human, fetal or non-fetal,child or adult, such as from a tissue or fluid, including blood, whichare obtainable through appropriate sample collection methods, such asbiopsy, blood collection or otherwise. Eukaryotic cells can be providedas is in a sample or can be cell lines that are cultivated in vitro.Differences in cell types also include cellular origin, distinct surfacemarkers, sizes, morphologies and other physical and biologicalproperties.

A “cell fragment” refers to a portion of a cell, such as cellorganelles, including but not limited to nuclei, endoplasmic reticulum,mitochondria or Golgi apparatus. Cell fragments can include vesicles,such as inside out or outside out vesicles or mixtures thereofPreparations that include cell fragments can be made using methods knownin the art.

A “population of cells” refers to a sample that includes more than onecell or more than one type of cell. For example, a sample of blood froma subject is a population of white cells and red cells. A population ofcells can also include a sample including a plurality of substantiallyhomogeneous cells, such as obtained through cell culture methods for acontinuous cell lines.

A “population of cell fragments” refers to a sample that includes morethan one cell fragment or more than one type of cell fragments. Forexample, a population of cell fragments can include mitochondria,nuclei, microsomes and portions of Golgi apparatus that can be formedupon cell lysis.

A “microparticle” is a structure of any shape and of any compositionthat is manipulatable by desired physical force(s). The microparticlesused in the methods could have a dimension from about 0.01 micron toabout ten centimeters. Preferably, the microparticles used in themethods have a dimension from about 0.1 micron to about several hundredmicrons. Such particles or microparticles can be comprised of anysuitable material, such as glass or ceramics, and/or one or morepolymers, such as, for example, nylon, polytetrafluoroethylene(Teflon™), polystyrene, polyacrylamide, sepaharose, agarose, cellulose,cellulose derivatives, or dextran, and/or can comprise metals. Examplesof microparticles include, but are not limited to, plastic particles,ceramic particles, carbon particles, polystyrene microbeads, glassbeads, magnetic beads, hollow glass spheres, metal particles, particlesof complex compositions, microfabricated free-standing microstructures,etc. The examples of microfabricated free-standing microstructures mayinclude those described in “Design of asynchronous dielectricmicromotors” by Hagedorn et al., in Journal of Electrostatics, Volume:33, Pages 159-185 (1994). Particles of complex compositions refer to theparticles that comprise or consists of multiple compositional elements,for example, a metallic sphere covered with a thin layer ofnon-conducting polymer film.

“A preparation of microparticles” is a composition that comprisesmicroparticles of one or more types and can optionally include at leastone other compound, molecule, structure, solution, reagent, particle, orchemical entity. For example, a preparation of microparticles can be asuspension of microparticles in a buffer, and can optionally includespecific binding members, enzymes, inert particles, surfactants,ligands, detergents, etc.

“Coupled” means bound. For example, a moiety can be coupled to amicroparticle by specific or nonspecific binding. As disclosed herein,the binding can be covalent or non-covalent, reversible or irreversible.

“Micro-scale structures” are structures integral to or attached on achip, wafer, or chamber that have characteristic dimensions of scale foruse in microfluidic applications ranging from about 0.1 micron to about20 mm. Example of micro-scale structures that can be on chips of thepresent invention are wells, channels, scaffolds, electrodes,electromagnetic units, or microfabricated pumps or valves.

A “particle positioning means” refers to a means that is capable ofmanipulating the position of a particle relative to the X—Y coordinatesor X—Y-Z coordinates of a biochip. Positions in the X—Y coordinates arein a plane. The Z coordinate is perpendicular to the plane. In oneaspect of the present invention, the X—Y coordinates are substantiallyperpendicular to gravity and the Z coordinate is substantially parallelto gravity. This need not be the case, however, particularly if thebiochip need not be level for operation or if a gravity free or gravityreduced environment is present. Several particle positioning means aredisclosed herein, such as but not limited to dielectric structures,dielectric focusing structures, quadrupole electrode structures,electrorotation structures, traveling wave dielectrophoresis structures,concentric electrode structures, spiral electrode structures, circularelectrode structures, square electrode structures, particle switchstructures, electromagnetic structures, DC electric field induced fluidmotion structure, acoustic structures, negative pressure structures andthe like. A “dielectric focusing structure” refers to a structure thatis on or within a biochip or a chamber that is capable of modulating theposition of a particle in the X—Y or X—Y-Z coordinates of a biochipusing dielectric forces or dielectrophoretic forces.

A “horizontal positioning means” refers to a particle positioning meansthat can position a particle in the X—Y coordinates of a biochip orchamber wherein the Z coordinate is substantially defined by gravity.

A “vertical positioning means” refers to a particle positioning meansthat can position a particle in the Z coordinate of a biochip or chamberwherein the Z coordinate is substantially defined by gravity.

A “quadrupole electrode structure” refers to a structure that includesfour electrodes arranged around a locus such as a hole, capillary orneedle on a biochip and is on or within a biochip or a chamber that iscapable of modulating the position of a particle in the X—Y or X—Y-Zcoordinates of a biochip using dielectrophoretic forces or dielectricforces generated by such quadrupole electrode structures.

An “electrorotation structure” refers to a structure that is on orwithin a biochip or a chamber that is capable of producing a rotatingelectric field in the X—Y or X—Y-Z coordinates that can rotate aparticle. Preferred electrorotation structures include a plurality ofelectrodes that are energized using phase offsets, such as 360/Ndegrees, where N represents the number of electrodes in theelectroroation structure (see generally U.S. patent application Ser. No.09/643,362 entitled “Apparatus and Method for High ThroughputElectrorotation Analysis” filed Aug. 22, 2000, naming Jing Cheng et al.as inventors). A rotating electrode structure can also producedielectrophoretic forces for positioning particles to certain locationsunder appropriate electric signal or excitation. For example, when N=4and electrorotation structure corresponds to a quadrupole electrodestructure.

A “traveling wave dielectrophoresis structure” refers to a structurethat is on or within a biochip or a chamber that is capable ofmodulating the position of a particle in the X—Y or X—Y-Z coordinates ofa biochip using traveling wave dielectrophoretic forces (see generallyU.S. patent application Ser. No. 09/686,737 filed Oct. 10, 2000, to Xu,Wang, Cheng, Yang and Wu; and U.S. application Ser. No. 09/678,263,entitled “Apparatus for Switching and Manipulating Particles and Methodsof Use Thereof” filed on Oct. 3, 2000 and naming as inventors XiaoboWang, Weiping Yang, Junquan Xu, Jing Cheng, and Lei Wu).

A “concentric circular electrode structure” refers to a structure havingmultiple concentric circular electrodes that are on or within a biochipor a chamber that is capable of modulating the position of a particle inthe X—Y or X—Y-Z coordinates of a biochip using dielectrophoreticforces.

A “spiral electrode structure” refers to a structure having multipleparallel spiral electrode elements that is on or within a biochip or achamber that is capable of modulating the position of a particle in theX—Y or X—Y-Z coordinates of a biochip using dielectric forces.

A “square spiral electrode structure” refers to a structure havingmultiple parallel square spiral electrode elements that are on or withina biochip or a chamber that is capable of modulating the position of aparticle in the X—Y or X—Y-Z coordinates of a biochip usingdielectrophoretic or traveling wave dielectrophoretic forces.

A “particle switch structure” refers to a structure that is on or withina biochip or a chamber that is capable of transporting particles andswitching the motion direction of a particle or particles in the X—Y orX—Y-Z coordinates of a biochip. The particle switch structure canmodulate the direction that a particle takes based on the physicalproperties of the particle or at the will of a programmer or operator(see, generally U.S. application Ser. No. 09/678,263, entitled“Apparatus for Switching and Manipulating Particles and Methods of UseThereof” filed on Oct. 3, 2000 and naming as inventors Xiaobo Wang,Weiping Yang, Junquan Xu, Jing Cheng, and Lei Wu.

An “electromagnetic structure” refers to a structure that is on orwithin a biochip or a chamber that is capable of modulating the positionof a particle in the X—Y or X—Y-Z coordinates of a biochip usingelectromagnetic forces. See generally U.S. patent application Ser. No.09/685,410 filed Oct. 10, 2000, to Wu, Wang, Cheng, Yang, Zhou, Liu andXu and WO 00/54882 published Sep. 21, 2000 to Zhou, Liu, Chen, Chen,Wang, Liu, Tan and Xu.

A “DC electric field induced fluid motion structure” refers to astructure that is on or within a biochip or a chamber that is capable ofmodulating the position of a particle in the X—Y or X—Y-Z coordinates ofa biochip using DC electric field that produces a fluidic motion.

An “electroosomosis structure” refers to a structure that is on orwithin a biochip or a chamber that is capable of modulating the positionof a particle in the X—Y or X—Y-Z coordinates of a biochip usingelectroosmotic forces. Preferably, an electroosmosis structure canmodulate the positioning of a particle such as a cell or fragmentthereof with an ion transport measuring means such that the particle'sseal (or the particle's sealing resistance) with such ion transportmeasuring means is increased.

An “acoustic structure” refers to a structure that is on or within abiochip or a chamber that is capable of modulating the position of aparticle in the X—Y or X—Y-Z coordinates of a biochip using acousticforces. In one aspect of the present invention, the acoustic forces aretransmitted directly or indirectly through an aqueous solution tomodulate the positioning of a particle. Preferably, an acousticstructure can modulate the positioning of a particle such as a cell orfragment thereof with an ion transport measuring means such that theparticle's seal with such ion transport measuring means is increased.

A “negative pressure structure” refers to a structure that is on orwithin a biochip or a chamber that is capable of modulating the positionof a particle in the X—Y or X—Y-Z coordinates of a biochip usingnegative pressure forces, such as those generated through the use ofpumps or the like. Preferably, a negative pressure structure canmodulate the positioning of a particle such as a cell or fragmentthereof with an ion transport measuring means such that the particle'sseal with such ion transport measuring means is increased.

“Dielectrophoresis” is the movement of polarized particles in electricalfields of nonuniform strength. There are generally two types ofdielectrophoresis, positive dielectrophoresis and negativedielectrophoresis. In positive dielectrophoresis, particles are moved bydielectrophoretic forces toward the strong field regions. In negativedielectrophoresis, particles are moved by dielectrophoretic forcestoward weak field regions. Whether moieties exhibit positive or negativedielectrophoresis depends on whether particles are more or lesspolarizable than the surrounding medium.

A “dielectrophoretic force” is the force that acts on a polarizableparticle in an AC electrical field of non-uniform strength. Thedielectrophoretic force {right arrow over (F)}_(DEP) acting on aparticle of radius r subjected to a non-uniform electrical field can begiven, under the dipole approximation, by:{right arrow over (F)}_(DEP)=2πε_(m)r³χ_(DEP)∇E² _(rms)where E_(rms) is the RMS value of the field strength, the symbol ∇ isthe symbol for gradient-operation, ε_(m) is the dielectric permittivityof the medium, and χ_(DEP) is the particle polarization factor, givenby:${\chi_{DEP} = {{Re}\left( \frac{ɛ_{p}^{*} - ɛ_{m}^{*}}{ɛ_{p}^{*} + {2ɛ_{m}^{*}}} \right)}},$“Re” refers to the real part of the “complex number”. The symbolε*_(x)=ε_(x)−jσ_(x)/2πf is the complex permittivity (of the particlex=p, and the medium x=m) and j=√{square root over (−1)}. The parametersε_(p) and σ_(p) are the effective permittivity and conductivity of theparticle, respectively. These parameters may be frequency dependent. Forexample, a typical biological cell will have frequency dependent,effective conductivity and permittivity, at least, because of cytoplasmmembrane polarization. Particles such as biological cells havingdifferent dielectric properties (as defined by permittivity andconductivity) will experience different dielectrophoretic forces. Thedielectrophoretic force in the above equation refers to the simpledipole approximation results. However, the dielectrophoretic forceutilized in this application generally refers to the force generated bynon-uniform electric fields and is not limited by the dipolesimplification. The above equation for the dielectrophoretic force canalso be written as{right arrow over (F)}_(DEP)=2πε_(m)r³χ_(DEP)V²∇p(x,y,z)where p(x,y,z) is the square-field distribution for a unit-voltageexcitation (Voltage V=1 V) on the electrodes, V is the applied voltage.

“Traveling-wave dielectrophoretic (TW-DEP) force” refers to the forcethat is generated on particles or molecules due to a traveling-waveelectric field. An ideal traveling-wave field is characterized by thedistribution of the phase values of AC electric field components, beinga linear function of the position of the particle. In this case thetraveling wave dielectrophoretic force {right arrow over (F)}_(TW-DEP)on a particle of radius r subjected to a traveling wave electrical fieldE=E cos(2π(ft−z/λ₀){right arrow over (a)}_(x) (i.e., a x-direction fieldis traveling along the z-direction) is given, again, under the dipoleapproximation, by${\overset{\rightharpoonup}{F}}_{{TW} - {DEP}} = {{- \frac{4\pi^{2}ɛ_{m}}{\lambda_{0}}}r^{3}\zeta_{{TW} - {DEP}}{E^{2} \cdot {\overset{\rightarrow}{a}}_{z}}}$where E is the magnitude of the field strength, ε_(m) is the dielectricpermittivity of the medium. ζ_(TW-DEP) is the particle polarizationfactor, given by${\zeta_{{TW} - {DEP}} = {{Im}\left( \frac{ɛ_{p}^{*} - ɛ_{m}^{*}}{ɛ_{p}^{*} + {2ɛ_{m}^{*}}} \right)}},$“Im” refers to the imaginary part of the “complex number”. The symbolε*_(x)=ε_(x)−jσ_(x)/2πf is the complex permittivity (of the particlex=p, and the medium x=m). The parameters ε_(p) and ε_(p) are theeffective permittivity and conductivity of the particle, respectively.These parameters may be frequency dependent.

A traveling wave electric field can be established by applyingappropriate AC signals to the microelectrodes appropriately arranged ona chip. For generating a traveling-wave-electric field, it is necessaryto apply at least three types of electrical signals each having adifferent phase value. An example to produce a traveling wave electricfield is to use four phase-quadrature signals (0, 90, 180 and 270degrees) to energize four linear, parallel electrodes patterned on thechip surfaces. Such four electrodes may be used to form a basic,repeating unit. Depending on the applications, there may be more thantwo such units that are located next to each other. This will produce atraveling-electric field in the spaces above or near the electrodes. Aslong as electrode elements are arranged following certain spatiallysequential orders, applying phase-sequenced signals will result inestablishing traveling electrical fields in the region close to theelectrodes.

“Electric field pattern” refers to the field distribution in space or ina region of interest. An electric field pattern is determined by manyparameters, including the frequency of the field, the magnitude of thefield, the magnitude distribution of the field, and the distribution ofthe phase values of the field components, the geometry of the electrodestructures that produce the electric field, and the frequency and/ormagnitude modulation of the field.

“Dielectric properties” of a particle are properties that determine, atleast in part, the response of a particle to an electric field. Thedielectric properties of a particle include the effective electricconductivity of a particle and the effective electric permittivity of aparticle. For a particle of homogeneous composition, for example, apolystyrene bead, the effective conductivity and effective permittivityare independent of the frequency of the electric field at least for awide frequency range (e.g. between 1 Hz to 100 MHz). Particles that havea homogeneous bulk composition may have net surface charges. When suchcharged particles are suspended in a medium, electrical double layersmay form at the particle/medium interfaces. Externally applied electricfield may interact with the electrical double layers, causing changes inthe effective conductivity and effective permittivity of the particles.The interactions between the applied field and the electrical doublelayers are generally frequency dependent. Thus, the effectiveconductivity and effective permittivity of such particles may befrequency dependent. For moieties of nonhomogeneous composition, forexample, a cell, the effective conductivity and effective permittivityare values that take into account the effective conductivities andeffective permittivities of both the membrane and internal portion ofthe cell, and can vary with the frequency of the electric field. Inaddition, the dielectrophoretic force experience by a particle in anelectric field is dependent on its size; therefore, the overall size ofparticle is herein considered to be a dielectric property of a particle.Properties of a particle that contribute to its dielectric propertiesinclude but are not limited to the net charge on a particle; thecomposition of a particle (including the distribution of chemical groupsor moieties on, within, or throughout a particle); size of a particle;surface configuration of a particle; surface charge of a particle; andthe conformation of a particle. Particles can be of any appropriateshape, such as geometric or non-geometric shapes. For example, particlescan be spheres, non-spherical, rough, smooth, have sharp edges, besquare, oblong or the like.

“Magnetic forces” refer to the forces acting on a particle due to theapplication of a magnetic field. In general, particles have to bemagnetic or paramagnetic when sufficient magnetic forces are needed tomanipulate particles. For a typical magnetic particle made ofsuper-paramagnetic material, when the particle is subjected to amagnetic field {right arrow over (B)}, a magnetic dipole {right arrowover (μ)} is induced in the particle $\begin{matrix}{{\overset{\rightarrow}{\mu} = {{V_{p}\left( {\chi_{p} - \chi_{m}} \right)}\frac{\overset{\rightarrow}{B}}{\mu_{m}}}},} \\{= {{V_{p}\left( {\chi_{p} - \chi_{m}} \right)}{\overset{\rightarrow}{H}}_{m}}}\end{matrix}$where V_(p) is the particle volume, χ_(p) and χ_(m) are the volumesusceptibility of the particle and its surrounding medium, μ_(m) is themagnetic permeability of medium, {right arrow over (H)}_(m) is themagnetic field strength. The magnetic force {right arrow over(F)}_(magnetic) acting on the particle is determined, under the dipoleapproximation, by the magnetic dipole moment and the magnetic fieldgradient:{right arrow over (F)}_(magnetic)=−0.5 V_(p)(χ_(p)−χ_(m)){right arrowover (H)}_(m)●∇{right arrow over (B)}_(m),where the symbols “●” and “∇” refer to dot-product and gradientoperations, respectively. Whether there is magnetic force acting on aparticle depends on the difference in the volume susceptibility betweenthe particle and its surrounding medium. Typically, particles aresuspended in a liquid, non-magnetic medium (the volume susceptibility isclose to zero) thus it is necessary to utilize magnetic particles (itsvolume susceptibility is much larger than zero). The particle velocityv_(particle) under the balance between magnetic force and viscous dragis given by:$v_{particle} = \frac{{\overset{\rightarrow}{F}}_{magnetic}}{6\pi\quad r\quad\eta_{m}}$where r is the particle radius and η_(m) is the viscosity of thesurrounding medium.

As used herein, “manipulation” refers to moving or processing of theparticles, which results in one-, two- or three-dimensional movement ofthe particle, in a chip format, whether within a single chip or betweenor among multiple chips. Non-limiting examples of the manipulationsinclude transportation, focusing, enrichment, concentration,aggregation, trapping, repulsion, levitation, separation, isolation orlinear or other directed motion of the particles. For effectivemanipulation, the binding partner and the physical force used in themethod should be compatible. For example, binding partner such asmicroparticles that can be bound with particles, having magneticproperties are preferably used with magnetic force. Similarly, bindingpartners having certain dielectric properties, for example, plasticparticles, polystyrene microbeads, are preferably used withdielectrophoretic force.

A “sample” is any sample from which particles are to be separated oranalyzed. A sample can be from any source, such as an organism, group oforganisms from the same or different species, from the environment, suchas from a body of water or from the soil, or from a food source or anindustrial source. A sample can be an unprocessed or a processed sample.A sample can be a gas, a liquid, or a semi-solid, and can be a solutionor a suspension. A sample can be an extract, for example a liquidextract of a soil or food sample, an extract of a throat or genitalswab, or an extract of a fecal sample. Samples are can include cells ora population of cells. The population of cells can be a mixture ofdifferent cells or a population of the same cell or cell type, such as aclonal population of cells. Cells can be derived from a biologicalsample from a subject, such as a fluid, tissue or organ sample. In thecase of tissues or organs, cells in tissues or organs can be isolated orseparated from the structure of the tissue or organ using known methods,such as teasing, rinsing, washing, passing through a grating andtreatment with proteases. Samples of any tissue or organ can be used,including mesodermally derived, endodermally derived or ectodermallyderived cells. Particularly preferred types of cells are from the heartand blood. Cells include but are not limited to suspensions of cells,cultured cell lines, recombinant cells, infected cells, eukaryoticcells, prokaryotic cells, infected with a virus, having a phenotypeinherited or acquired, cells having a pathological status including aspecific pathological status or complexed with biological ornon-biological entities.

“Separation” is a process in which one or more components of a sample isspatially separated from one or more other components of a sample or aprocess to spatially redistribute particles within a sample such as amixture of particles, such as a mixture of cells. A separation can beperformed such that one or more particles is translocated to one or moreareas of a separation apparatus and at least some of the remainingcomponents are translocated away from the area or areas where the one ormore particles are translocated to and/or retained in, or in which oneor more particles is retained in one or more areas and at least some orthe remaining components are removed from the area or areas.Alternatively, one or more components of a sample can be translocated toand/or retained in one or more areas and one or more particles can beremoved from the area or areas. It is also possible to cause one or moreparticles to be translocated to one or more areas and one or moremoieties of interest or one or more components of a sample to betranslocated to one or more other areas. Separations can be achievedthrough the use of physical, chemical, electrical, or magnetic forces.Examples of forces that can be used in separations include but are notlimited to gravity, mass flow, dielectrophoretic forces, traveling-wavedielectrophoretic forces, and electromagnetic forces.

“Capture” is a type of separation in which one or more particles isretained in one or more areas of a chip. In the methods of the presentapplication, a capture can be performed when physical forces such asdielectrophoretic forces or electromagnetic forces are acted on theparticle and direct the particle to one or more areas of a chip.

An “assay” is a test performed on a sample or a component of a sample.An assay can test for the presence of a component, the amount orconcentration of a component, the composition of a component, theactivity of a component, the electrical properties of an ion transportprotein, etc. Assays that can be performed in conjunction with thecompositions and methods of the present invention include, but notlimited to, biochemical assays, binding assays, cellular assays, geneticassays, ion transport assay, gene expression assays and proteinexpression assays.

A “binding assay” is an assay that tests for the presence or theconcentration of an entity by detecting binding of the entity to aspecific binding member, or an assay that tests the ability of an entityto bind another entity, or tests the binding affinity of one entity foranother entity. An entity can be an organic or inorganic molecule, amolecular complex that comprises, organic, inorganic, or a combinationof organic and inorganic compounds, an organelle, a virus, or a cell.Binding assays can use detectable labels or signal generating systemsthat give rise to detectable signals in the presence of the boundentity. Standard binding assays include those that rely on nucleic acidhybridization to detect specific nucleic acid sequences, those that relyon antibody binding to entities, and those that rely on ligands bindingto receptors.

A “biochemical assay” is an assay that tests for the composition of orthe presence, concentration, or activity of one or more components of asample.

A “cellular assay” is an assay that tests for or with a cellularprocess, such as, but not limited to, a metabolic activity, a catabolicactivity, an ion transport function or property, an intracellularsignaling activity, a receptor-linked signaling activity, atranscriptional activity, a translational activity, or a secretoryactivity.

An “ion transport assay” is an assay useful for determining iontransport functions or properties and testing for the abilities andproperties of chemical entities to alter ion transport functions.Preferred ion transport assays include electrophysiology-based methodswhich include, but are not limited to patch clamp recording, whole cellrecording, perforated patch or whole cell recording, vesicle recording,outside out and inside out recording, single channel recording,artificial membrane channel recording, voltage gated ion transportrecording, ligand gated ion transport recording, stretch activated(fluid flow or osmotic) ion transport recording, and recordings onenergy requiring ion transporters (such as ATP), non energy requiringtransporters, and channels formed by toxins such a scorpion toxins,viruses, and the like. See, generally Neher and Sakman, ScientificAmerican 266:44-51 (1992); Sakmann and Heher, Ann. Rev. Physiol.46:455-472 (1984); Cahalan and Neher, Methods in Enzymology 207:3-14(1992); Levis and Rae, Methods in Enzymology 207:14-66 (1992); Armstrongand Gilly, Methods in Enzymology 207:100-122 (1992); Heinmann and Conti,Methods in Enzymology 207:131-148 (1992); Bean, Methods in Enzymology207:181-193 (1992); Leim et al., Neurosurgery 36:382-392 (1995); Lester,Ann. Rev. Physiol 53:477-496 (1991); Hamill and McBride, Ann. Rev.Physiol 59:621-631 (1997); Bustamante and Varranda, Brazilian Journal31:333-354 (1998); Martinez-Pardon and Ferrus, Current Topics inDevelopmental Biol. 36:303-312 (1998); Herness, Physiology and Behavior69:17-27 (2000); Aston-Jones and Siggins,www.acnp.org/GA/GN40100005/CH005.html (Feb. 8, 2001); U.S. Pat. No.6,117,291; U.S. Pat. No. 6,107,066; U.S. Pat. No. 5,840,041 and U.S.Pat. No. 5,661,035; Boulton et al., Patch-Clamp Applications andProtocols, Neuromethods V. 26 (1995), Humana Press, New Jersey;Ashcroft, Ion Channels and Disease, Cannelopathies, Academic Press, SanDiego (2000); Sakmann and Neher, Single Channel Recording, secondedition, Plenuim Press, New York (1995) and Soria and Cena, Ion ChannelPharmacology, Oxford University Press, New York (1998), each of which isincorporated by reference herein in their entirety.

An “electrical seal” refers to a high-resistance engagement between aparticle such as a cell or cell membrane and an ion transport measuringmeans, such as a hole, capillary or needle of a chip or device of thepresent invention. Preferred resistance of such an electrical seal isbetween about 1 mega ohm and about 100 giga ohms, but that need not bethe case. Generally, a large resistance results in decreased noise inthe recording signals. For specific types of ion channels (withdifferent magnitude of recording current) appropriate electric sealingin terms of mega ohms or giga ohms can be used.

An “acid” includes acid and acidic compounds and solutions that have apH of less than 7 under conditions of use.

A “base” includes base and basic compounds and solutions that have a pHof greater than 7 under conditions of use.

“More electronegative” means having a higher density of negative charge.In the methods of the present invention, a chip or ion transportmeasuring means that is more electronegative has a higher density ofnegative surface charge.

An “electrolyte bridge” is a liquid (such as a solution) or a solid(such as an agar salt bridge) conductive connection with at least onecomponent of the electrolyte bridge being an electrolyte so that thebridge can pass current with no or low resistance.

A “ligand gated ion transport” refers to ion transporters such as ligandgated ion channels, including extracellular ligand gated ion channelsand intracellular ligand gated ion channels, whose activity or functionis activated or modulated by the binding of a ligand. The activity orfunction of ligand gated ion transports can be detected by measuringvoltage or current in response to ligands or test chemicals. Examplesinclude but are not limited to GABA_(A), strychnine-sensitive glycine,nicotinic acetylcholine (Ach), ionotropic glutamate (iGlu), and5-hydroxytryptamine₃ (5-HT₃) receptors.

A “voltage gated ion transport” refers to ion transporters such asvoltage gated ion channels whose activity or function is activated ormodulated by voltage. The activity or function of voltage gated iontransports can be detected by measuring voltage or current in responseto different commanding currents or voltages respectively. Examplesinclude but are not limited to voltage dependent Na⁺ channels.

“Perforated” patch clamp refers to the use of perforation agents such asbut not limited to nystatin or amphotericin B to form pores orperforations that are preferably ion-conducting, which allows for themeasurement of current, including whole cell current.

An “electrode” is a structure of highly electrically conductivematerial. A highly conductive material is a material with conductivitygreater than that of surrounding structures or materials. Suitablehighly electrically conductive materials include metals, such as gold,chromium, platinum, aluminum, and the like, and can also includenonmetals, such as carbon, conductive liquids and conductive polymers.An electrode can be any shape, such as rectangular, circular,castellated, etc. Electrodes can also comprise doped semi-conductors,where a semi-conducting material is mixed with small amounts of other“impurity” materials. For example, phosphorous-doped silicon may be usedas conductive materials for forming electrodes.

A “channel” is a structure with a lower surface and at least two wallsthat extend upward from the lower surface of the channel, and in whichthe length of two opposite walls is greater than the distance betweenthe two opposite walls. A channel therefore allows for flow of a fluidalong its internal length. A channel can be covered (a “tunnel”) oropen.

“Continuous flow” means that fluid is pumped or injected into a chamberof the present invention continuously during the separation process.This allows for components of a sample that are not selectively retainedon a chip to be flushed out of the chamber during the separationprocess.

“Binding partner” refers to any substances that both bind to themoieties with desired affinity or specificity and are manipulatable withthe desired physical force(s). Non-limiting examples of the bindingpartners include cells, cellular organelles, viruses, particles,microparticles or an aggregate or complex thereof, or an aggregate orcomplex of molecules.

A “specific binding member” is one of two different molecules having anarea on the surface or in a cavity that specifically binds to and isthereby defined as complementary with a particular spatial and polarorganization of the other molecule. A specific binding member can be amember of an immunological pair such as antigen-antibody, can bebiotin-avidin or biotin streptavidin, ligand-receptor, nucleic acidduplexes, IgG-protein A, DNA-DNA, DNA-RNA, RNA-RNA, and the like.

A “nucleic acid molecule” is a polynucleotide. A nucleic acid moleculecan be DNA, RNA, or a combination of both. A nucleic acid molecule canalso include sugars other than ribose and deoxyribose incorporated intothe backbone, and thus can be other than DNA or RNA. A nucleic acid cancomprise nucleobases that are naturally occurring or that do not occurin nature, such as xanthine, derivatives of nucleobases, such as2-aminoadenine, and the like. A nucleic acid molecule of the presentinvention can have linkages other than phosphodiester linkages. Anucleic acid molecule of the present invention can be a peptide nucleicacid molecule, in which nucleobases are linked to a peptide backbone. Anucleic acid molecule can be of any length, and can be single-stranded,double-stranded, or triple-stranded, or any combination thereof. Theabove described nucleic acid molecules can be made by a biologicalprocess or chemical synthesis or a combination thereof.

A “detectable label” is a compound or molecule that can be detected, orthat can generate readout, such as fluorescence, radioactivity, color,chemiluminescence or other readouts known in the art or later developed.Such labels can be, but are not limited to, photometric, colorimetric,radioactive or morphological such as changes of cell morphology that aredetectable, such as by optical methods. The readouts can be based onfluorescence, such as by fluorescent labels, such as but not limited to,Cy-3, Cy-5, phycoerythrin, phycocyanin, allophycocyanin, FITC,rhodamine, or lanthanides; and by fluorescent proteins such as, but notlimited to, green fluorescent protein (GFP). The readout can be based onenzymatic activity, such as, but not limited to, the activity ofbeta-galactosidase, beta-lactamase, horseradish peroxidase, alkalinephosphatase, or luciferase. The readout can be based on radioisotopes(such as ³³P, ³H, ¹⁴C, ³⁵S, ¹²⁵I, ³²P or ¹³¹I). A label optionally canbe a base with modified mass, such as, for example, pyrimidines modifiedat the C5 position or purines modified at the N7 position. Massmodifying groups can be, for examples, halogen, ether or polyether,alkyl, ester or polyester, or of the general type XR, wherein X is alinking group and R is a mass-modifying group. One of skill in the artwill recognize that there are numerous possibilities formass-modifications useful in modifying nucleic acid molecules andoligonucleotides, including those described in Oligonucleotides andAnalogues: A Practical Approach, Eckstein, ed. (1991) and inPCT/US94/00193.

A “signal producing system” may have one or more components, at leastone component usually being a labeled binding member. The signalproducing system includes all of the reagents required to produce orenhance a measurable signal including signal producing means capable ofinteracting with a label to produce a signal. The signal producingsystem provides a signal detectable by external means, often bymeasurement of a change in the wavelength of light absorption oremission. A signal producing system can include a chromophoric substrateand enzyme, where chromophoric substrates are enzymatically converted todyes which absorb light in the ultraviolet or visible region, phosphorsor fluorophores. However, a signal producing system can also provide adetectable signal that can be based on radioactivity or other detectablesignals.

The signal producing system can include at least one catalyst, usuallyat least one enzyme, and can include at least one substrate, and mayinclude two or more catalysts and a plurality of substrates, and mayinclude a combination of enzymes, where the substrate of one enzyme isthe product of the other enzyme. The operation of the signal producingsystem is to produce a product that provides a detectable signal at thepredetermined site, related to the presence of label at thepredetermined site.

In order to have a detectable signal, it may be desirable to providemeans for amplifying the signal produced by the presence of the label atthe predetermined site. Therefore, it will usually be preferable for thelabel to be a catalyst or luminescent compound or radioisotope, mostpreferably a catalyst. Preferably, catalysts are enzymes and coenzymesthat can produce a multiplicity of signal generating molecules from asingle label. An enzyme or coenzyme can be employed which provides thedesired amplification by producing a product, which absorbs light, forexample, a dye, or emits light upon irradiation, for example, afluorophore. Alternatively, the catalytic reaction can lead to directlight emission, for example, chemiluminescence. A large number ofenzymes and coenzymes for providing such products are indicated in U.S.Pat. No. 4,275,149 and U.S. Pat. No. 4,318,980, which disclosures areincorporated herein by reference. A wide variety of non-enzymaticcatalysts that may be employed are found in U.S. Pat. No. 4,160,645,issued Jul. 10, 1979, the appropriate portions of which are incorporatedherein by reference.

The product of the enzyme reaction will usually be a dye or fluorophore.A large number of illustrative fluorophores are indicated in U.S. Pat.No. 4,275,149, which is incorporated herein by reference.

Other technical terms used herein have their ordinary meaning in the artthat they are used, as exemplified by a variety of technicaldictionaries.

Introduction

The present invention recognizes that using direct detection methods todetermine an ion transport function or property, such as patch-clamps,is preferable to using indirect detection methods, such asfluorescence-based detection systems. The present invention providesbiochips and methods of use that allow for the direct detection of oneor more ion transport functions or properties using chips and devicesthat can allow for automated detection of one or more ion transportfunctions or properties. These biochips and methods of use thereof areparticularly appropriate for automating the detection of ion transportfunctions or properties, particularly for screening purposes.

As a non-limiting introduction to the breath of the present invention,the present invention includes several general and useful aspects,including:

-   -   1) a biochip device for ion transport measurement that comprises        at least one upper chamber piece and at least one biochip that        comprises at least one ion transport measuring means. The device        can comprise one or more conduits that provide an electrolyte        bridge to at least one electrode.    -   2) a biochip ion transport measuring device having one or more        flow-through lower chambers.    -   3) a biochip devices adapted for a microscope stage.    -   4) methods of making an upper piece for a biochip device for ion        transport measurement.    -   5) methods for making chips comprising ion transport measurement        holes using laser drilling techniques.    -   6) devices that include an inverted chip for ion transport        measurement.    -   7) methods of treating ion transport measuring chips to enhance        their sealing properties.    -   8) a method to measure surface energy, such as on the surface of        a chemically-treated ion transport measurement biochip.    -   9) substrates, biochips, cartridges, apparatuses, and/or devices        comprising ion transport measuring means with enhanced electric        seal properties.    -   10) methods for storing the substrates, biochips, cartridges,        apparatuses, and/or devices comprising ion transport measuring        means with enhanced electrical seal properties.    -   11) methods for shipping the substrates, biochips, cartridges,        apparatuses, and/or devices comprising ion transport measuring        means with enhanced electrical seal properties.    -   12) methods for assembling devices and cartridges of the present        invention using UV adhesives.    -   13) a method of producing ion transport measuring chips by        fabricating them as detachable units of a large sheet.    -   14) a method of producing high density ion transport measuring        chips.    -   15) a biochip device for ion transport measurement comprising        fluidic channel upper and lower chambers.    -   16) methods of preparing cells for ion transport measurement.    -   17) a software program logic that controls a pressure profile to        direct an ion transport measurement apparatus to achieve and        maintain a high-resistance electrical seal.    -   18) an ion channel chip having chemical surface modification on        plastics and methods of use. Preferably, the plastic surface is        modified to improve the chip's ability for form tight seals for        ion transport measurement. Preferably, plastic surfaces can be        plasma etched, which makes the surface clean and creates        chemical functional groups that can be used for further chemical        reactions and/or polymerizations to provide functionalities on        the surface. These modifications are preferably made after holes        are provided on the chip, such as through laser drilling. A        variety of formats of holes can be provided, preferably in a        standard format or footprint, such as between 1 and 1536 holes        or more on a chip. These chips can be used in methods of        determining ion channel activities, including high throughput        methods.    -   19) a bilayer-bilayer junction for forming tight seals and        method of use. A lipid bilayer is provided to cover a hole in a        chip used for ion transport measurement. The lipid is probably        attached to a surface of a chip, such as through covalent or        non-covalent attachment. Preferably, head groups of a lipid        bilayer are attached to the surface. The surface thus has a        negative charge that would promote the formation of a tight seal        for use in ion transport measurement.    -   20) a method of making a pre-assembled ion transport measurement        cartridge and method of use. In this aspect of the present        invention, a chip without a hole is provided in a cartridge,        such as described herein or in the applications incorporated by        reference. The assembled cartridge is then oriented for drilling        of the chip to form holes for use in ion transport measurement.        The resulting cartridge with a chip with holes can then be        treated for surface modification to promote the formation of        tight seals for ion transport measurement. The treatment can be        any appropriate treatment, preferably those described herein,        the applications incorporated by reference herein, or as known        or described in the art. These cartridges can be used to perform        ion transport measurements as described herein, the applications        incorporated by reference herein, or as known or described in        the art.    -   21) a method of coating a plastic chip with glass and method of        use. Generally, a plastic chip is coated with glass. The glass        coated chip is then laser drilled or wet-etched to form holes        useful for determination of ion channel activity.    -   22) a method of protecting fragile devices and parts by packing        and shipping in a fluid such as water. Many of the chips, either        alone or in combination with cartridge structures, can be        fragile and difficult to transport without breakage. By        packaging, storing and shipping fragile structures in a fluid        such as water, breakage can be reduced.    -   23) a method laser beam splitting and method of use to laser        drill holes in ion channel chips and methods of use. The method        utilizes a homogenizer to obtain a “top hat” power profile from        a laser beam source. The laser beam is then optionally masked at        this point in order to provide one or more profiles for laser        drilling a substrate for use as an ion channel detection        structure. The laser beam is then passed through a beam splitter        to make two or more laser beamlets. The laser beamlets are then        optionally masked to provide one or more profiles for laser        drilling a substrate for use as an ion channel detection        structure. Either the first, second or both of the masking steps        can be used. The laser beamlets are then focused through a        single or multiple lenses onto a work-piece. The work-piece is        in one or more parts and is laser drilled to form structures for        use in ion channel detection methods.    -   24) a method of making glass more readily wet etched, ion        channel chips made using this method, and methods of use thereof        Generally, the invention converts glass to a form that is more        readily wet etched by interaction with a laser in the UV range.        The glass that is exposed to such laser light changes structure        to become more readily wet etched. The methods are preferably        used for making counter-bores, thinnings and through-holes.        Glass appropriate for such procedures is an amorphous glass or        glass ceramic that it thermal sensitive. One example of such        glass is commercially available from Invenions or Schott, and        has a product name of Foturan.    -   25) methods for bonding glass to glass and products produced by        such methods. The method makes use of flat surfaces of glass        that an operator is desires to bond together. The two pieces of        glass can be untreated or treated independently with acid and/or        base. The resulting laminate is heated to bond the two pieces of        glass together. Alternatively, the two pieces of glass can be        untreated or treated independently with acid and/or base. NaSiO4        powder is placed between the two pieces of glass. The laminate        is heated to melt the NaSiO4 and thus bond the two pieces of        glass together.    -   26) an in situ method of making a cartridge for use in ion        transport measurement and methods of use. A chip with at least        one hole for use in ion transport measurement is provided to an        instrument used to make ion transport measurement. The chip is        placed such that gaskets are provided on the top surface of the        chip and the bottom surface of the chip to form top and bottom        chambers. The chambers leave the hole or holes exposed for use        in ion transport measurement. A cross section of such a        configuration wherein the gaskets engage a chip with holes is        provided in FIG. 53.    -   27) a gasket for use in ion transport measurement and methods of        use thereof. One aspect of the present invention is depicted in        FIG. 54. A gasket is provided to engage a chip for use in ion        transport measurement. The gasket can be made of any appropriate        material that can form a water tight or water resistant seal        with a chip. Preferred materials include, but are not limited        to, plastics, rubbers, silicones, gels and the like. The gasket        includes a variety of structures to engage a chip and form        chambers for use in ion transport measurement. These structures        include 0-rings, vacuum holes and vents.    -   28) a system for automated processing of chips for use in ion        transport measurement. One aspect of the invention is provided        in FIG. 55. Briefly, a chip is provided in a treatment storage        solution. A treated chip is picked up by a structure having        negative pressure to engage a chip. The chip is passed over        structures providing negative and positive pressure to dry the        chip. The chip is then further dried in a structure having        negative pressure and optionally moving the chip from side to        side. The chip is then removed using the structure having        negative pressure and assembled into a cartridge, a storage        structure or an instrument for use in ion transport measurement.    -   29) a device that can hold chips for methods of treating chips        and/or for storage of chips. One aspect of the present invention        is provided in FIG. 56. The structure has a hinged top structure        that engages a bottom structure, wherein the top structure and        bottom structure are reversibly engaged using a clip. The bottom        structure includes separate structures to hold or otherwise        engage chips of the present invention at a location for chips.        In the structure set forth in FIG. 56, the location for chip is        configured for long thin chips. Other configurations for other        chip sizes can be readily designed and made within the scope and        spirit of the present invention. In operation, the top structure        is lifted away from the bottom structure via the hinge. One or        more chips are placed individually in the location for chips.        Preferably, only one chip is placed in one location for chip.        The top structure engages the bottom structure and is reversibly        held in place by the clip. The structure with the chips can be        used to store chips or be used to hold chips during treatment.        Alternatively, the structure with chips can be provided to        instrumentation and/or robotics for movement of chips. The        structure can be made of any appropriate material, such as but        not limited to plastic, glass, rubber and the like. The        structure is preferably made of plastic and is preferably made        using injection molding.

These aspects of the invention, as well as others described herein, canbe achieved by using the methods, articles of manufacture andcompositions of matter described herein. To gain a full appreciation ofthe scope of the present invention, it will be further recognized thatvarious aspects of the present invention can be combined to makedesirable embodiments of the invention.

Device for Ion Transport Measurement

The present invention comprises devices for ion transport measurementand components of ion transport measuring devices that reduce the costsof manufacture and use and are efficient and convenient to use. Thedevices of the present invention are also designed for maximumversatility, providing for different assay formats within the same basicdesign.

In some aspects, the present invention contemplates devices andapparatuses that have parts that are manufactured separately and can beassembled to form ion transport measuring devices that have at leastone, and preferably multiple, ion transport measuring units, each ofwhich comprises an upper chamber, at least a portion of a biochip thatcomprises an ion transport measuring means that connects the upperchamber with a lower chamber. These devices comprising ion channelmeasuring units can be assembled before the assay procedure, and piecesthat make up the device can be reversibly or irreversibly attached toone another. In many preferred aspects of the present invention, one ormore portions of an ion transport measuring device will be permanent andreusable (for example, at least a portion of a lower chamber; one ormore electrodes) and one or more parts of a device can be removed froman apparatus and can be disposable (for example, a chip comprising iontransport measuring means; one or more upper chambers designed tocontain cells). In some aspects of the present invention, a devicecomprising one or more upper chamber pieces and at least one biochip(called a cartridge) are single-use and disposable, and lower chamberpieces, one or more electrodes, and platforms or lower base pieces arereusable. In these aspects, upper chamber pieces and biochips can bereversibly or irreversibly attached to one another and during use of thedevice or apparatus, these attached upper chamber/biochip devices can bereversibly attached to or contacted with lower chamber pieces, conduits,or electrodes.

In one embodiment, the present invention contemplates an ion transportmeasuring device in the form of a cartridge that comprises an upperchamber piece that comprises at least one well that is open at its upperand lower ends, and a biochip that comprises at least one ion transportmeasuring means. The upper well or wells are in register with the iontransport measuring means, providing independent upper chambers each incontact with a single ion transport measuring means. Preferably abiochip that is part of an ion transport measuring device of the presentinvention comprises one or more holes used as ion transport measuringmeans, and an upper chamber piece comprises multiple upper chambers suchthat upper chambers are in register with ion transport measuring meansof the chip.

An upper chamber piece can be made of any suitable material, but forease of manufacturing is preferably made of a moldable plastic, such as,for example, polyallomer, polyethylene, polyimide, polypropylene,polystyrene, polycarbonate, cylco-olefin polymer, polyphenyleneether/PPO, NORYL®, ZEONOR® or composite polymers. A cross section of onedesign of an upper base piece is shown in FIG. 1(D). A chip can besealed to the lower surface of the upper base piece to form a cartridge

The chip used in a device of the present invention is preferably a chipthat comprises ion transport measuring means in the form of holes.Methods of fabricating such chips, including methods of fabricating iontransport measuring holes in chips, are disclosed herein. The chips arepreferably chemically treated to have enhanced sealing properties.Methods of treating ion transport measuring chips with basic solutionsto enhance their ability to form electrical seals with particles such ascells are also disclosed herein. A preferred cartridge, known as theSEALCHIP™, comprises a chip with enhanced electrical sealing propertiesthat is reversibly or irreversibly attached to an upper chamber piece.

An upper chamber piece can optionally comprise one or more electrodes.An upper chamber piece that comprises multiple upper chambers cancomprise multiple electrodes, where each well contacts an independentelectrode (such as, for example, independent recording electrodes), orcan have a single electrode that contacts all of the upper chambers ofthe device (which can be, for example, a reference electrode). Where theupper chamber piece does not comprise one or more electrodes, the upperchamber piece can optionally be used as part of an apparatus for iontransport measurement in which one or more electrodes can be introducedinto one or more upper chambers (such as, for example, introduced via aconduit that can be connected to or can be inserted into one or morechambers). In an alternative configuration, conduits connected with orintroduced into one or more upper chambers can, during the use of theapparatus, be filled with a measuring solution and provide electrolytebridges to one or more electrodes.

A cartridge comprising an upper chamber piece and at least one biochipcomprising one or more ion transport measuring means can be assembledwith a lower chamber piece that comprises at least a portion of at leastone lower chamber. The cartridge can be assembled with a lower chamberpiece that comprises at least a portion of a single lower chamber, suchas a dish, tray, or channel that provides a common lower chamber for iontransport measuring means that connect to separate upper chambers. Inone embodiment, a lower chamber piece can be in the form of a gasketthat seals around the bottom of the biochip that when sealed against alower chamber base piece provides an inner space as a lower chamber.

Alternatively, the device can be assembled with a lower chamber piecethat comprises at least a portion of more than one lower chamber. Inthis case, each individual lower chamber would connect with a singleupper chamber via the ion transport measuring hole in the biochip.

Preferred embodiments encompass devices that comprise multiple iontransport measuring units, comprising an upper chamber piece thatcomprises at least two upper chambers that are open at both their upperand lower ends, a chip that comprises at least two ion transportmeasuring means in the form of holes through the chip that are inregister with the upper chambers, and a lower chamber piece thatcomprises at least a portion of at least two lower chambers that are inregister with the ion transport measuring means and upper chambers.

Lower chamber pieces that comprise at least a portion of more than onelower chamber can be provided in a variety of designs. Lower chamberpieces can comprise complete chamber units, or can comprise all or aportion of the walls of the multiple chamber units, such that when thelower chamber piece is fixed to or pressed against the lower side of abiochip and pressed down on a platform or lower chamber base piece, thelower chamber piece forms the walls and the platform or lower chamberbase piece forms the bottoms of the lower chambers.

For example, a device for measuring ion transport function or activitycan be a multiple unit device that comprises an upper chamber piecehaving multiple upper chambers in the form of wells that are open atboth the top and bottom, a chip attached to the upper chamber piece,where the chip comprises multiple holes for ion transport measurementthat are spaced such that when the device is assembled each upperchamber is over a hole, and a lower chamber piece that can be held orfastened against the lower side of the chip, in which the lower chamberpiece comprises multiple openings that fit over the biochip holes toform lower chambers.

In this preferred embodiment, the lower chamber piece can comprise atleast one compressible plastic or polymer on its upper surface that canform a seal with the bottom of the biochip. The lower chamber piece canalso comprise at least one compressible polymer as a gasket on its uppersurface that can form a seal with a platform or a lower base piece. Whenthe device is positioned on a lower base piece or platform so that thelower chamber piece is pressed against the lower base piece or platform,the lower base piece or platform forms the bottom of the lower chambers.Mechanical pressure can provide a seal between the biochip and the lowerchamber piece, and between the lower chamber piece and the platform.Clamps can optionally be employed to hold the seal. The compressibleplastic or polymer can comprise rubber, a plastic, or an elastomer, suchas for example, polydimethylsiloxane (PDMS), silicon polyether urethane,polyester elastomer, polyether ester elastomer, olefinic elastomer,polyurethane elastomer, polyether block amide, or styrenic elastomer.Preferably, in cases where the compressible plastic or polymer contactscells, the compressible plastic or polymer is made of a biocompatiblematerial, such as PDMS. Portions of the lower chamber piece that do notform a gasket can be of any suitable material, including polymers,metals, and ceramics. Portions of the lower chamber piece that contactmeasuring solutions preferably comprise materials that are not affectedby electrical current (such as nonmetals).

For example, one preferred design of a device for ion transportmeasurement comprises an upper chamber piece, a chip comprising iontransport measuring holes, a lower chamber piece, and a lower base piecein the form of a platform. The chip has been chemically treated,preferably with at least one base, to enhance its sealing properties.The lower chambers that are formed by a lower chamber piece thatcomprises an aluminum frame having a PDMS gasket on its upper surfacethat fits over the lower surface of a chip. PDMS is also used to coatthe inner surfaces of the holes that form the lower chambers, and isalso used as a gasket on the bottom of the lower chamber piece. Thelower chambers can be filled with a solution while the device is held ininverted orientation prior to positioning the device on the platform.During use of the device, mechanical pressure holds the lower chamberpiece against the chip and against the platform.

The lower base piece can optionally comprise one or more electrodes. Forexample, separate individual electrodes can be fabricated on or attachedto the platform so that separate lower chambers of the device haveindependent electrodes that can be attached to independent circuits andused as patch clamp recording electrodes. In an alternative design, theplatform can comprise a common lower chamber with a reference electrode,or a common electrode that can contact all of the lower chambers of thedevice (optionally through separate electrode extensions that meet acommon connector outside of the chambers) and can be used as a referenceelectrode.

The lower base piece can optionally comprise or engage one or moreconduits connected to tubing that can allow for the flow of fluids intoand out of individual lower chambers. In this type of design the lowerchambers can be filled with a measuring solution (such as anintracellular solution) after the gasket is positioned on a lower basepiece. The conduits can also be used for the exchange of solutionsduring the use of the device. For example, solutions containing testcompounds, ionophores, inhibitors, drugs, different concentrations orcombinations of ions or compounds, etc., can be delivered into and outof a chamber during ion transport measuring assays. At least some of theconduits or tubing can optionally comprise or lead to electrodes (suchas, but not limited to, recording electrodes).

In one preferred design, a lower chamber piece having a platform thatforms the bottom of lower chambers comprises conduits that engage eachlower chamber from one side (one per chamber), and conduits that engageeach lower chamber from the opposite side. Conduits on one side of thelower chamber piece can be used for introducing solutions, such as“intracellular solutions” that can optionally comprise test compounds,into the chambers, and conduits on the opposite side of the lowerchamber piece can be used for flushing solutions and/or air bubbles outof the lower chambers. At least one set of the conduits (such as, forexample, the inflow conduits) comprises wire electrodes that areindependently connected (with respect to other ion transport measuringunits) to a signal amplifier and used for ion transport activityrecording.

Devices such as those described herein can be part of apparatuses thatalso comprise patch clamp signal amplifiers and conduits, fluiddispensing means, pumps, electrodes, or other components. Theapparatuses are preferably mechanized, for automated fluid dispensing orpumping, pressure generation for sealing of particles, and ion transportrecording. The apparatuses can be part of a biochip system for iontransport measurement, in which software controls the automatedfunctions.

An Ion Channel Measurment Device Adapted for Microscopes

The present invention also includes a lower chamber base piece for usein a device for ion transport measurement that can optionally be usedindependently of a larger automated apparatus and can be used to observecells and particles within the device using an inverted microscope. Atleast a portion of the lower chamber base piece is transparent, andpreferably the lower chamber base piece comprises at least two conduitsthat are capable of transferring fluid from one surface of the lowerchamber platform to another surface of the base piece in a flow-throughmanner. As part of a device for ion transport measurement, the basepiece forms a bottom surface of lower chambers. The conduits that extendthrough the base piece allow for fluids, and liquid solutions inparticular, to be delivered in and out of lower chambers of iontransport measuring devices.

The two or more conduits go through the base piece, with one opening onone surface of the base piece, and the other opening on a differentsurface of the base piece. In preferred embodiments of the presentinvention, the conduits extend from a side of the base piece essentiallyhorizontally toward the center, and then turn or curve upward to end inan opening on the top surface of the base piece. The side opening can bethe site where the conduit connects with tubing connected to solutionreservoirs, pressure sources, and/or electrodes, and the top opening ofthe conduits is the site where the conduit opens into a lower chamber.Each lower chamber of an ion transport measuring device preferably isconnected to two such conduits, and the conduits can provide forsolutions to be delivered into and washed out of a lower chamber.

The part or parts of a lower chamber base piece that will form thebottom of one or more lower chambers of an ion transport measuringdevice is preferably made of a transparent material that is impermeableto aqueous liquids so that cells or particles inside an ion transportmeasuring unit are visible using an inverted microscope. Although not arequirement of the present invention, to simplify manufacture of thebase piece, the entire base piece (with the exception of separateattachments such as connectors, pins, screws, etc.) is preferably madeof a single material by molding or machining. Glass and transparentpolymers are preferred materials, with transparent polymers such aspolycarbonate and polystyrene having the advantage of easiermanufacture.

The conduits are molded or drilled through the base piece, and can befitted with connectors. (Connectors can comprise glass, polymers,plastics, ceramics, or metals.) The connectors can be connected totubing that can be used to provide in-flow and outflow of solutions to alower chamber of an ion transport measuring unit.

The conduits can also be used to deliver pressure to the lower chamberand to an ion transport measuring hole of a chip exposed to the chamber.Pressure can be generated, for example, by a pump or a pressure sourceconnected to the tubing that will be filled with an appropriate solutionin at least the segment connecting the lower chamber. Preferably thepressure is regulatable and can be used for purging air bubbles and orother blocking micro-particles in the ion transport measuring hole, celland particle positioning, sealing, and optionally, membrane rupture ofan attached cell when carrying out ion transport measurement procedures.

In preferred embodiments, the conduits, or tubes leading to theconduits, can also comprise electrodes. For example, a wire electrodecan be threaded through tubing that is connected to a conduit of a basepiece. The wire electrode can optionally extend through the conduit tothe upper surface of the base piece (which will be the lower surface ofa lower chamber of an ion transport measuring unit).

In the alternative, the base piece can comprise one or more electrodeson its upper surface. Electrodes fabricated or attached to the uppersurface of the base piece can be connected through leads to connectorson the outer edge of the base piece, and the connectors can be connectedto a patch clamp amplifier.

In preferred aspects of the present invention, a lower chamber basepiece is designed to fit a base plate that is adapted to fit the stageof a microscope, such as an inverted light microscope. The dimensionscan be altered to fit a microscope of choice, such as, for example, aninverted light microscope sold by Leica, Nikon, Olympus, Zeiss, or othercompanies.

A base plate can be made of any suitable material, such as glass,plastics, polymers, ceramics, or metals. Metals, such as but not limitedto stainless steel, are preferred, because metal materials have highmechanical strength needed during pressure sealing of the lower chamber.A metal base plate can also, together with a grounded microscope stage,form an electrical noise shield around a lower chamber piece fitted tothe base plate.

The base plate can be carved on the top side to catch any fluids thatmay leak or spill and prevent the contamination of the microscope withthe fluids. Preferably, the base plate is sealed around the lowerchamber base piece, for example, with silicone glue, silicone grease,Vaseline, etc.

The base plate is preferably drilled and tapped so as to provide amounting point for the lower chamber base piece and for a clamp that canhold additional components of the ion transport measuring devicetogether (for example, gasket, chip, upper chamber piece) to form theupper and lower chambers of ion transport measuring units. The baseplate is designed to hold an ion transport measuring device within a fewmillimeters of the level of the top of the microscope stage so as toensure that the chip function may be monitored within the focal range ofthe microscope. FIG. 2 illustrates the design of a base plate as adaptedfor a Nikon Microscope.

In preferred aspects of the present invention, a lower chamber basepiece is designed to form the bottom of more than one lower chamber ofan ion transport measuring device. Preferably, a lower chamber basepiece is designed to form the bottoms of all the lower chambers of anion transport measuring device that comprises at least two ion transportmeasuring units, more preferably at least three ion transport measuringunits, and more preferably yet, at least six ion transport measuringunits. In a preferred embodiment described in detail in Example 5, alower chamber base piece forms the bottom of 16 lower chambers of a 16unit device. In many cases (as illustrated in the example) multiplelower chambers will be arranged linearly in a row, but this is not arequirement of the present invention.

Thus, in preferred embodiments of the present invention, a lower chamberbase piece will comprise multiple conduits, two for each lower chamberthat will occur in the ion transport measuring device (a first conduitfor inflow of solutions, and a second conduit for outflow of solutions).A cross-sectional view of a chamber of one design of a lower base pieceis shown in FIG. 3. One of each pair of conduits that leads to a singlechamber of an ion transport measuring device can optionally contain orcontact an electrode.

The present invention also includes devices for ion transportmeasurement that include a lower chamber base piece of the presentinvention. Such apparatuses include: a lower chamber base piece thatcomprises at least two conduits; where at least a portion of the lowerchamber base piece is transparent; a chip comprising at least one iontransport measuring hole; at least one gasket that fits between thelower chamber base piece and the biochip and comprises at least onehole, such that the gasket forms the walls of the one or more lowerchambers and seals the lower chamber base piece to the chip such that alower chamber formed by the gasket comprises a lower surface having theopenings of two conduits, and an upper surface comprising a portion of achip having a single ion transport measuring hole; and an upper chamberpiece that comprises at least one chamber that attaches to said chip.Drawings of one preferred design of the present invention withflow-through lower chambers are provided as FIG. 4.

Preferably, tubing is attached to “out” openings of the conduits, andpreferably at least one electrode is within one of the two tubes thatengages a conduit that leads to a single lower chamber, or an electrodeis in electrical contact with the solution in one or the two tubes thatengages a conduit that leads to a single lower chamber. FIGS. 5-10.

Upper chambers of such devices can comprise electrodes. Such electrodescan be fabricated, positioned, or attached on a surface of an upperchamber, such as those described in a later section of this applicationon two-piece molding of upper chambers, or can be provided as within atube or part of a tube that can be placed inside the upper chamber (suchas a tube that delivers solutions or cell suspensions). Preferably,electrodes of upper chambers are connected as a common referenceelectrode, but this is not a requirement of the present invention. It isalso possible for each upper well to have an individual (recording)electrode, and to have the electrodes of the lower chambers connected asa common reference electrode.

The upper piece can optionally comprise one or more electrodes. In somepreferred embodiments, the upper piece comprises a common referenceelectrode that contacts all of the wells. In other preferredembodiments, an electrode is not within or attached to the upper piece,but an electrode can be brought into electrical contact with an upperchamber by way of a conduit that comprises an electrode or can providean electrolyte solution bridge to an electrode. Electrodes that areconnected through electrolyte bridges can be recording electrodes, butin most preferred embodiments are reference electrodes.

The present invention also encompasses compositions and devices thatincorporate novel elements of the compositions and devices describedherein, including: transparent platform beneath lower chambers,base-plate for microscope stage, bottom chamber flow through tubing,reference or recording electrodes outside of upper or lower chambers andconnected to chamber(s) through electrolyte bridge, reference orrecording electrodes introduced into tubing attached to upper or lowerchambers, manufacture procedures and features for enhancing efficiencyor accuracy of manufacture, tapering of upper chamber wells, geometry ofholes drilled into chips, counterbore drilling of holes in chips,treatment of chips to enhance electrical sealing of particles such ascells, etc.

Method of making an Upper Chamber Piece of a Device for Ion TransportMeasurement

In ion transport measuring devices contemplated by the presentinvention, an upper chamber is designed to contain the cells orparticles on which ion transport measurements are to be performed. Inthese embodiments, an upper chamber of an ion transport measuring devicecan comprise or engage at least a portion of an electrode used tomonitor ion transport activity. In the alternative, an upper chamber,when filled with an ion transport measuring solution, can be broughtinto electrical contact with at least a portion of an electrode. Forexample, an electrode (such as, but not limited to, a metal wire) can beinserted into the well so that electrical current from the electrodewould be transmitted through the conductive measuring solution.Alternatively, a tube that comprises a measuring solution (or otherwiseconductive solution) that contains or contacts an electrode or a portionthereof can be put in contact with the upper chamber solution. In thelatter case, the electrode (or a portion thereof) need not be within theupper chamber at all, as long as it is electrically connected to theinner part of the upper chamber conductive solution (electrolytebridge).

Typically, an upper chamber electrode will be a reference electrode,although this need not be the case. In cases in which upper chamberelectrodes are reference electrodes, electrode extensions or electrolytebridges that contact individual upper chambers can be connected with oneanother either outside or inside the upper chamber piece.

In many of the devices of the present invention, an upper chamber piececomprises at least one upper chambers in the form of a well. Preferably,an upper chamber piece comprises multiple upper chambers or wells thatallow several ion transport assays to be performed simultaneously. Theupper chamber piece can optionally comprise one or more electrodes. Thepresent invention provides methods of making upper chamber pieces thatincrease the efficiency and reduce the cost of making devices thatmeasure ion transport activity of cells and particles.

Two-Piece Molding followed by Electrode Insertion

In one aspect of the present invention, an upper chamber piece thatcomprises one or more wells is made in two pieces, an upper well portionpiece and a well hole portion piece, and the well hole portion piece hasa groove into which a wire electrode can fit. An upper well portionpiece comprises the upper portion of one or more wells. The upper wellportions are open at both ends. The well hole piece comprises one ormore well holes that will form the bottom portion of the one or morewells. A well hole is, in effect, the lower portion of a well and canhave different dimensions (height, diameter, and taper angle) than theupper well portion. The well holes are also open at their upper andlower ends. The well holes have an upper diameter that is equal orsmaller than the diameter of the lower opening of the upper wellportion. When the upper well portion piece is attached on top of thewell hole piece, the upper well portions are aligned over the well holesto form upper chambers (wells) that have well holes at their lower end.An example of this arrangement (upper well portion piece having upperportions of wells and lower well piece having well holes) is depicted inFIG. 11. After manufacturing the upper well portion piece and the wellhole piece, a wire electrode is inserted into the groove of the wellhole piece, and then the upper well portion piece is attached, via, forexample ultrasonic welding, to the well hole piece to form an upperchamber piece comprising one or more wells, each of which is in contactwith a portion of a wire electrode.

The method includes: molding a well hole portion piece of an upperchamber piece of an ion transport measuring device, wherein said wellhole portion piece comprises: at least one well hole, and a groove thatextends longitudinally from one end of the well hole portion piecetoward the opposite end of the well hole portion piece, such that thegroove contacts the one or more well holes; molding an upper wellportion piece of an upper chamber piece that comprises at least oneupper well; inserting a wire electrode into the groove of the well holeportion piece; and attaching the upper well portion piece to the wellhole portion piece to form an upper chamber piece that comprises one ormore wells, such that the wire electrode is exposed to the interior ofsaid one or more wells.

In this embodiment, the upper piece is made from one or more plasticsand comprises wells that are open at either end, and each well contactsor contains a portion of a common electrode that can be used as areference electrode in ion transport measuring assays. This method ofmanufacture is particularly suited embodiments where the upper piececomprises multiple wells (at least two) that will contact a commonelectrode, and wells are arranged linearly in a row. However, this isnot a requirement of the present invention, and the principle oftwo-piece molding and wire insertion can be adapted to the manufactureof device components in which multiple wells or chambers that will sharea common electrode are arranged in different geometries. In suchembodiments, the path of the groove can be designed such that itcontacts all of the wells or chambers that are intended to be in contactwith the electrode. This includes embodiments where there are multiplerows of wells or chambers, arrangement of wells or chambers inconcentric circles or spirals as well as other arrangements of wells orchambers.

It is also possible to adapt the methods of the present invention todesigns in which one or more wells are to be contacted by one electrodeand one or more other wells are to be contacted by a differentelectrode. It is also possible that one well be contacted with more thatone electrode. In such cases, the well hole portion piece will comprisemore than one continuous groove such that more than one wire electrodecan be inserted into the lower well portion piece.

Injection molding or compression molding techniques as they are known inthe art can be used to make the well hole portion piece and the upperwell portion piece. In the methods of the present invention, the upperwell portion piece comprises an upper portion of at least one well orchamber and the well hole portion piece comprises a lower portion of atleast one well or chamber, such that when the upper well portion pieceis attached to the well hole portion piece, the two pieces together format least one upper well or upper chamber. The well hole portion piececomprises at least one groove whose diameter corresponds to that of awire electrode, and the groove contacts the well holes. Preferably, thewell hole portion piece comprises a well hole whose upper diameter isequal to or smaller than the lower diameter of the upper portion of thewell that is part of the upper well portion piece. Thus, in preferredembodiments, the well hole portion piece will have a top surface aroundthe upper diameter of the well hole (see FIG. 11), that, when lookingdown into a well after the entire top chamber piece is assembled,appears as a ledge around the top of the well hole. The groove can be inthis top surface or ledge. In this way the wire electrode can be easilyinserted into the groove, and its placement on this “ledge” ensures thatit will be exposed to the interior of the well after attachment of theupper well portion piece.

The wire is easily inserted into the groove of the lower well portionpiece, as the groove is totally accessible prior to attachment the upperand lower portion pieces.

After insertion of the wire electrode, the upper well portion piece andwell hole portion piece are fused together to form a complete upperchamber piece. Any glues appropriate to the materials and applicationsof the devices can be used for this purpose. UV glues and otherfast-curing glues are preferred for mass production of the upper chamberpieces, although slow-cure glues can also be used for mass production ifa high capacity production process is used. Ultrasonic welding,pressuring, heating, or other bonding methods can also be used.

Upper Chamber Pieces and Devices

The present invention includes upper chamber pieces that are made usingthe methods of the present invention, and devices that comprise suchpieces. Such pieces and devices can comprise wells or chambers that areopen or closed at one or both ends, can comprise other components, suchas, but not limited to, membranes, microstructures, ports (optionallywith attached conduits), fluidic channels, particles positioning means,specific binding members, polymers, etc., and are not limited to use asion transport measuring devices. In fact, the same design andmanufacturing principles can be used to fabricate pieces that comprisewells or chambers that need not function as “upper” pieces of devices orapparatuses. Two-piece molding, wire insertion, and attachment of twopieces can be used to make devices or components of devices thatcomprise wells or chambers regardless of whether the components,chambers, or wells, can be considered “upper”.

Plastics that can be used in the manufacture of upper and lower piecesinclude, but are not limited to polyallomer, polypropylene, polystyrene,polycarbonate, cyclo-olefin polymer, polyimide, paralene, PDMS,polyphenylene ether/PPO, Noryl®, Zeonor®, etc. A very large number andvariety of moldable plastics and their properties are known. Forexample, a database of materials, including plastics and polymers usefulin the present invention, can be found at http://www.matweb.com.

Electrodes can comprise conductive materials such as metals that can beshaped into wires. Various metals, including aluminum, chromium, copper,gold, nickel, palladium, platinum, silver, steel, and tin can be used aselectrode materials. For electrodes used in ion channel measurement,wires made of silver or other metal halides are preferred, such asAg/AgCl wires.

The design and dimensions of the upper and lower well pieces, as well asthe dimension of the upper wells and lower wells, can vary according tothe preferences of the user and are not limiting to the presentinvention.

Preferred Embodiments: Upper Chamber Pieces and Devices

In preferred embodiments of the present invention, the upper chamberpiece comprises one or more upper wells that can function as the upperchambers of ion transport measuring units of ion transport measuringdevices. Preferably, an upper chamber piece of the present inventioncomprises more than one upper well, and more preferably more than twoupper wells. Even more preferably, an upper chamber piece comprises sixor more upper wells, each of which can be a part of an ion transportmeasuring unit of an ion transport measuring device, where all of thesix or more upper wells of the manufactured upper chamber piece contacta portion of a common wire electrode that extends along the upperchamber piece. FIGS. 5 and 6 depict a preferred upper chamber piece ofthe present invention that comprises sixteen wells.

The wells of an upper chamber piece that can be part of an ion transportmeasuring device preferably can hold a volume of between about 5microliters and about 5 milliliters, more preferably between about 10microliters and about 2 milliliters, and more preferably yet betweenabout 25 microliters and about 1 milliliter. The depth, or height of awell can vary from about 1 to 25 millimeters, and more preferably willbe from about 2 milliliters to about 10 milliliters in depth. Inpreferred embodiments of the present invention in which an upper wellportion and a lower well portion together make up the well, the upperwell portion is preferably from about 1 to about 25 milliliters indepth, and the lower well is preferably from about 100 microns to about10 milliliters in depth.

A low cell or particle density is often preferred for attaining a highsuccess rate when using the ion channel measuring device describedherein. In order to reduce the cell or particle density required foroptimal cell or particle landing to the recording apertures, it isdesirable to have an accurate means for delivering the cells orparticles to the recording aperture. For a more accurate delivery ofcells or particles to the recording aperture, the upper chamber well canhave one or more tapered walls, The walls can be contoured such that thecells or particles, when delivered to the upper chamber well wall (suchas by robotic dispenser), are directed to the recording aperture.

In these preferred embodiments, the shape of the well can vary, and canbe irregular or regular, and in many cases will be generally circular oroval at its circumference. In a preferred embodiment of the presentinvention depicted in FIG. 11 and FIG. 1, the wells of the upper chamberpiece are horseshoe-shaped, and at least a portion of the sides of thewells are tapered. FIG. 1 c, for example, shows that the wall of thewell corresponding to the rounded end of the horseshoe shape taperstoward the bottom of the upper portion of the well. In other preferredembodiments, the walls along entire well taper toward the bottom of theupper portion of the well. In these preferred embodiments, the diameterof a well at its upper end will generally be from about 2 millimeter toabout 10 millimeters.

In some preferred embodiments of the present invention the angle of thetaper of a portion of the walls of the well or the entire well walls(the difference from vertical) is from about one degree to about 80degrees. More preferably, the angle of the taper of the well walls isbetween about 5 degrees and 60 degrees from vertical. The taper canextend down the full height of the well, or the well can be tapered foronly a portion of its height. The upper well portion can optionally betapered, or the well hole can optionally be tapered, or both the upperwell portion and the lower well portion can be tapered. Where both aretapered, the tapering need not be to the same degree or extend aroundthe well to the same extent.

Molding of Single Upper Chamber Piece around Electrode

In another aspect of the present invention, an upper chamber piece withat least one wire electrode can be manufactured as a single piece bymolding an upper piece around a wire electrode. In this case, the moldhas a means for positioning the wire electrode such that the upperchamber piece that includes the wells can be molded around it. Themethod includes: positioning an electrode in a mold; and injectionmolding an upper chamber piece using the mold such that the electrodecontacts one or more wells of the upper chamber piece. The electrode canbe positioned in any of a number of ways, for example it can extendthrough the mold and be held by apertures that it is threaded through oneither end of the mold.

The injection molded upper chamber piece can comprise one or more wellsor upper chambers, preferably two or more, more preferably six or morewells. The wells can be of any dimension of size, and can comprise awell hole within the well as described in the previous section.

Molding of Single Upper Piece without Electrode

In yet another aspect of the present invention, an upper chamber piececan be manufactured without an electrode. In this case, an upper chamberpiece with a desirable number of wells is injection molded using asuitable plastic, such as, but not limited to, polyallomer,polypropylene, polystyrene, polycarbonate, polyimide, paralene, PDMS,cyclo-olefin polymer, polyphenylene ether/PPO, Noryl®, or Zeonor®.

When the upper chamber piece is integrated into a device for iontransport measurement, electrodes (for example, metal wires) can beinserted into the wells. Such electrodes are preferably referenceelectrodes and are preferably connected outside the chambers, butinserted electrodes can also be recording electrodes connectedseparately to a power source/signal amplifier.

In a preferred embodiment of the present invention, an electrodeconnection can be provided by a conduit that can be introduced into theupper chambers during use of the device. The conduit can comprise anelectrode, or, when the conduit is filled with a conductive solution,can be in electrical contact with an electrode. When both the upperchamber and the conduit contain a conductive solution (such as ameasuring solution), the upper chamber is in electrical contact with theelectrode through the “electrolyte bridge” of solution provided by theconduit.

Insert Molding of Glass Chip

In yet another embodiment, a pre-diced glass chip is insert-moldedtogether with an upper chamber piece to make a one-piece cartridge. Inthis process, a glass chip is inserted into a mold, and the upperchamber piece is molded around the glass chip such that it forms thebottoms of upper chambers of the upper chamber piece. Laser drilling ofthe recording apertures is done after the molding process, and then thecartridge is chemically treated to enhance its electrical sealingproperties. In this embodiment, materials that can be treated with acidand base (such as, for example, NORYL® and ZEONOR®) are used for theconstruction of the cartridge other than the biochip.

Additional Features

In some preferred embodiments of the present invention, the upperchamber pieces of the present invention or components of the upperchamber pieces of the present invention can have additional featuresthat can aid in the manufacture of upper chamber pieces or of iontransport measuring devices. One such feature is an alignment bump seenon the left of the chamber piece depicted in FIG. 1D. One or morealignment bumps on the lower surface of an upper chamber piece can beused during attachment of a chip that comprises ion transport measuringmeans to the upper chamber piece. Attachment of the chip and the upperchamber piece must occur such that every ion transport measuring hole inthe chip is aligned with a well hole. The alignment bump allows a personor machine assembling the device to detect the location where the edgeof the chip must be positioned.

Another useful feature for the manufacture of ion transport measuringdevices that can occur on upper chamber piece of the present inventionis a glue spillage groove. This allows for overflow of glue that is usedfor the attachment of a chip, such as a chip that comprises iontransport measuring means. The glue spillage groove is also shown asnotches in the bottom surface of the part shown in FIG. 1C.

Yet another optional feature useful in the manufacturing process of anupper chamber piece is the presence of sinkholes. Depicted in FIG. 1D,these sinkholes allow for the escape of gas during the molding process.

Methods of making a Chip Comprising Holes for Ion Transport Measurement

Fabrication of Ion Transport Measuring Holes in a Chip

The present invention also comprises methods of making chips comprisingholes for ion transport measurement. The method includes: providing asubstrate; laser drilling at least one counterbore in said substrate,and laser drilling at least one hole in said substrate. Preferably,laser drilling is done with sequential or simultaneous measurement ofthe glass thickness at the site of the pore.

For optimal quality ion transport recording, ion transport measurementchips comprising holes for ion transport measurement ideally should havea low hole resistance (Re) across the chip at the site of the hole. Forchips having multiple holes, it is also desirable to have a high degreeof uniformity of hole resistance from recording site to recording site.The hole resistance decreases with decreasing depth of the hole in thechip and widening of the exit hole (see FIGS. 12 and 13). A widertapering (greater angle from vertical) of the hole also decreases Re.

The methods of the present invention seek to reduce hole resistance byreducing hole depth. This is achieved by laser drilling holes in thinsubstrates, such as glass, quartz, silicon, silicon dioxide, or polymersubstrates. The graph provided in FIG. 13 illustrates that thinner chips(“K-configuration chips”) have a lower Re than those with greater holedepth. The graph provided in FIG. 12 illustrates that a decreasing holedepth and widening the exit hole decreases Ra.

A chip with shortened holes for ion transport measurement can be made bylaser drilling one or more counterbores into a glass chip, and thenlaser drilling a through hole through the one or more counterbores.While a wide counterbore is preferred for lower Re, increased width ofthe counterbore can weaken the chip. It is also difficult to control thedrilling of the counterbore as the bottom of the counterbore getsthinner and thinner. In addition, with increased (deeper) drilling, theperipheral areas of the counterbores tend to be deeper than the morecentral portions of the counterbore due to optical waveguide effects(this is sometimes called the “Batman Effect”). To avoid these problems,a second counterbore is laser drilled into the bottom of a firstcounterbore. This makes drilling to a greater depth easier control, andhas the effect of reducing the thickness of the chip in the vicinity ofthe through hole. Thus, synthesis of biochips for ion transportmeasurement can include laser drilling at least one counterbore througha substrate, and then drilling a through hole through the one or morecounterbores. Preferably two counterbores are laser drilled into asubstrate, such that a second counterbore is drilled through a firstcounterbore, that is, the counterbores are nested to form (along with athrough hole) a single hole structure. In some embodiments of thepresent invention, three, four, or more nested counterbores can bedrilled into a substrate prior to drilling a through hole through thecounterbores.

Control of the depth of laser drilling can be done by using a separatelaser device that can measure the thickness of the glass. In preferredaspects of this embodiment of the present invention, a measuring laseris used to measure the thickness of the substrate before or as it isbeing drilled, and the laser used for drilling can be regulated by thethickness of the remaining substrate at the bottom surface of thecounterbore. Laser-based measuring devices have been used for thedetermination of glass thickness to an accuracy of 0.1 micron. Such alaser measurement device is available from the Keyence Company. A laserbased measurement is made to determine the exact thickness of thesubstrate. This measurement determines the number of pulses to be usedby the drilling laser to drill a counterbore and thereby achieveuniformity of hole depth. To improve the consistency of through holedepth and hole resistance, the invention contemplates the integration ofa laser unit with an excimer laser drilling device, together withautomated control software.

In practice, a substrate made of glass, quartz, silicon, silicondioxide, polymers, or other substrates that preferably ranges inthickness from 5 to 1000 microns, and more preferably from 10 to 200microns, is provided. A first counterbore is laser drilled, where theentrance of the counterbore has a diameter from about 20 to about 200microns, preferably from about 40 to about 120 microns. The firstcounterbore can be drilled to a depth of from about 100 microns to thethickness of the substrate minus the through hole depth. Depending onthe thickness of the substrate and the number of counterbores that eachion transport measuring hole will have. Subsequent counterbores willhave a smaller diameter than the first counterbore, and can be of lesserdepth than the first counterbore. In general, after drilling of all ofthe counterbores that will be part of an ion transport measuring hole,the remaining thickness of the substrate that is to be drilled out toform the through hole (that is, the depth of the through hole) willrange from about 0.5 to about 200 microns, and preferably will rangefrom about 2 to about 50 microns, more preferably from about 5 to about30 microns. The diameter of the through hole can be from about 0.2 toabout 8 microns, and preferably will be from about 0.5 to about 5microns, and even more preferably, from about 0.5 to about 3 microns.

Counterbores can be tapered. Preferably, a counterbore is tapered at anangle ranging from about 1 degree to about 80 degrees from vertical, andmore preferably from about 3 degrees to about 45 degrees from vertical.Ion transport measuring holes comprising multiple counterbores can havedifferent taper angles for different counterbores.

Through holes can also be tapered. The angle of taper for a through holecan range from about 0 degree to about 75 degrees from vertical, andmore preferably, where a through hole is tapered, is from about 0 degreeto about 45 degrees from vertical. In general an exit hole of a throughhole will have a narrower diameter than an entrance hole, although thisis not a requirement of the present invention.

Inverted Chip

The present invention also includes methods of using chips comprisingion transport measuring holes that are in inverted orientation for iontransport measurement, that is, using chips in which the holes (or atleast a portion of the holes, such as a portion of the holes made by atleast one counterbore) have a negative taper.

The method comprises: assembling a device for ion transport measurementthat comprises: at least one upper chamber, wherein the one or moreupper chambers comprise or are in electrical contact with at least oneelectrode; at least one chip that comprises a measuring hole, whereinthe one or more chips are assembled in the device in invertedorientation; and at least one lower chamber, wherein the one or morelower chambers comprise or are in electrical contact with at least oneelectrode; connecting the electrodes with a power supply/signalamplifier; introducing at least one particle or at least one cell intoat least one upper chamber, and measuring ion transport activity of atleast one cell or at least one particle.

By “inverted orientation” is meant that, for a chip which ion transportmeasuring holes are made by drilling, the chip is positioned such thatthe side of the chip having the laser entrance hole opening is exposedto a chamber that will contain cells or particles, instead of the sidehaving the laser exit hole. This is contrary to what has previously beendone in the art. Thus, sealing of a cell or particles against the iontransport measuring hole occurs on the side of the chip opposite to theside that has smaller hole size (the “back side” of the chip).

The inverted chip orientation has several advantages. One advantage isthat the chip does not require a laser polishing step, since the laserdrilling performs this function as a “side-effect”. A second advantageis that sealing occurs with high efficiency due to the geometry of theparticle-chip interaction. Yet another advantage is that a stable low Racan be produced using larger holes (for example, from about 2 to about 5microns in diameter), due to the position at which break-in occursduring whole cell recording.

When one or counterbores are used to reduced the through hole depth, thethrough hole can be drilled from either the same direction as thecounterbores, or from the opposite direction to the counterbores. In theformer case, the chips is produced just like the “normal” chips areproduced, they are simply assembled up side down.

The present invention includes devices and apparatuses having chipscomprising ion transport measuring holes that are in invertedorientation, as well as methods of using chips comprising ion transportmeasuring holes that are in inverted orientation for ion transportmeasurement.

Methods of Treating Chips Comprising Ion Transport Measuring means toEnhance the Electrical Seal of a Particle

The present invention also includes methods of modifying an iontransport measuring means to enhance the electrical seal of a particleor membrane with the ion transport measuring means. Ion transportmeasuring means includes, as non-limiting examples, holes, apertures,capillaries, and needles. “Modifying an ion transport measuring means”means modifying at least a portion of the surface of a chip, substrate,coating, channel, or other structure that comprises or surrounds the iontransport measuring means. The modification may refer to the surfacesurrounding all or a portion of the ion transport measuring means. Forexample, a biochip of the present invention that comprises an iontransport measuring means can be modified on one or both surfaces (e.g.upper and lower surfaces) that surround an ion transport measuring hole,and the modification may or may not extend through all or a part of thesurface surrounding the portion of the hole that extends through thechip. Similarly, for capillaries, pipettes, or for channels or tubestructures that comprises ion transport measuring means (such asapertures), the inner surface, outer surface, or both, of the channel,tube, capillary, or pipette can be modified, and all or a portion of thesurface that surrounds the inner aperture and extends through thesubstrate (and optionally, coating) material can also be modified.

As used herein, “enhance the electrical seal”, “enhance the electricseal”, “enhance the electric sealing” or “enhance the electrical sealingproperties (of a chip or an ion transport measuring means)” meansincrease the resistance of an electrical seal that can be achieved usingan ion transport measuring means, increase the efficiency of obtaining ahigh resistance electrical seal (for example, reducing the timenecessary to obtain one or more high resistance electrical seals), orincreasing the probability of obtaining a high resistance electricalseal (for example, the number of high resistance seals obtained within agiven time period).

The method comprises: providing an ion transport measuring means andtreating the ion transport measuring means to enhance the electricalsealing properties of the ion transport measuring means. Preferably,treating an ion transport measuring means to enhance the electricalsealing properties results in a change in surface properties of the iontransport measuring means. The change in surface properties can be achange in surface texture, a change in surface cleanness, a change insurface composition such as ion composition, a change in surfaceadhesion properties, or a change in surface electric charge on thesurface of the ion transport measuring means. In some preferred aspectsof the present invention, a substrate or structure that comprises an iontransport measuring means is subjected to chemical treatment (forexample, treated in acid, and/or base for specified lengths of timeunder specified conditions). For example, treatment of a glass chipcomprising a hole through the chip as an ion transport measuring meanswith acid and/or base solutions may result in a cleaner and smoothersurface in terms of surface texture for the hole. In addition, treatinga surface of a biochip or fluidic channel that comprises an iontransport measuring means (such as a hole or aperture) or treating thesurface of a pipette or capillary with acid and/or base may alter thesurface composition, and/or modify surface hydrophobicity and/or changesurface charge density and/or surface charge polarity.

Preferably, the altered surface properties improve or facilitate a highresistance electric seal or high resistance electric sealing between thesurface-modified ion transport measuring means and a membranes orparticle. In preferred embodiments of the present invention in which theion transport measuring means are holes through one or more biochips,one or more biochips having ion transport measuring means with enhancedsealing properties (or, simply, a “biochip having enhanced sealingproperties”) preferably has a rate of at least 50% high resistancesealing, in which a seal of 1 Giga Ohm or greater occurs at 50% of theion transport measuring means takes place in under 2 minutes after acell lands on an ion transport measuring hole, and preferably within 10seconds, and more preferably, in 2 seconds or less. Preferably, forbiochips with enhanced sealing properties, a 1 Giga Ohm resistance sealis maintained for at least 3 seconds.

In practice, in preferred aspects of the present invention the methodcomprises providing an ion transport measuring means and treating theion transport measuring means with one or more of the following: heat, alaser, microwave radiation, high energy radiation, salts, reactivecompounds, oxidizing agents (for example, peroxide, oxygen plasma),acids, or bases. Preferably, an ion transport measuring means or astructure (as nonlimiting examples, a structure can be a substrate,chip, tube, or channel, any of which can optionally comprise a coating)that comprises at least one ion transport measuring means is treatedwith one or more agents to alter the surface properties of the iontransport measuring means to make at least a portion of the surface ofthe ion transport measuring means smoother, cleaner, or moreelectronegative.

An ion transport measuring means can be any ion transport measuringmeans, including a pipette, hole, aperture, or capillary. An aperturecan be any aperture, including an aperture in a channel, such as withinthe diameter of a channel (for example, a narrowing of a channel), inthe wall of a channel, or where a channel forms a junction with anotherchannel. (As used herein, “channel” also includes subchannels.) In somepreferred aspects of the present invention, the ion transport measuringmeans is on a biochip, on a planar structure, but the ion transportmeasuring means can also be on a non-planar structure.

The ion transport measuring means or surface surrounding the iontransport measuring means modified to enhance electrical sealing cancomprise any suitable material. Preferred materials include silica,glass, quartz, silicon, plastic materials, polydimethylsiloxane (PDMS),or oxygen plasma treated PDMS. In some preferred aspects of the presentinvention, the ion transport measuring means comprises SiOM surfacegroups, where M can be hydrogen or a metal, such as, for example, Na, K,Mg, Ca, etc. In such cases, the surface density of said SiOM surfacegroups (or oxidized SiOM groups (SiO⁻)) is preferably more than about1%, more preferably more than about 10%, and yet more preferably morethan about 30%. The SiOM group can be on a surface, for example, thatcomprises glass, for example quartz glass or borosilicate glass,thermally oxidized SiO₂ on silicon, deposited SiO₂, deposited glass,polydimethylsiloxane (PDMS), or oxygen plasma treated PDMS.

In preferred embodiments, the method comprises treating said iontransport measuring means with acid, base, salt solutions, oxygenplasma, or peroxide, by treating with radiation, by heating (forexample, baking or fire polishing) by laser polishing said ion transportmeasuring means, or by performing any combinations thereof.

An acid used for treating an ion transport measuring means can be anyacid, as nonlimiting examples, HCl, H₂SO₄, NaHSO₄, HSO₄, HNO₃, HF,H₃PO₄, HBr, HCOOH, or CH₃COOH can be. The acid can be of a concentrationabout 0.1 M or greater, and preferably is about 0.5 M or higher inconcentration, and more preferably greater than about 1 M inconcentration. Optimal concentrations for treating an ion transportmeasuring means to enhance its electrical sealing properties can bedetermined empirically. The ion transport measuring means can be placedin a solution of acid for any length of time, preferably for more thanone minute, and more preferably for more than about five minutes. Acidtreatment can be done under any non-frozen and non-boiling temperature,preferably at greater or equal than room temperature.

An ion transport measuring means can be treated with a base, such as abasic solution, that can comprise, as nonlimiting examples, NaOH, KOH,Ba(OH)₂, LiOH, CsOH, or Ca(OH)₂. The basic solution can be of aconcentration of about 0.01 M or greater, and preferably is greater thanabout 0.05 M, and more preferably greater than about 0.1 M inconcentration. Optimal concentrations for treating an ion transportmeasuring means to enhance its electrical sealing properties can bedetermined empirically (see examples). The ion transport measuring meanscan be placed in a solution of base for any length of time, preferablyfor more than one minute, and more preferably for more than about fiveminutes. Base treatment can be done under any non-frozen and non-boilingtemperature, preferably at greater or equal than room temperature.

An ion transport measuring means can be treated with a salt, such as ametal salt solution, that can comprise, as nonlimiting examples, NaCl,KCl, BaCl₂, LiCl, CsCl, Na₂SO₄, NaNO₃, or CaCl, etc. The salt solutioncan be of a concentration of about 0.1 M or greater, and preferably isgreater than about 0.5 M, and more preferably greater than about 1 M inconcentration. Optimal concentrations for treating an ion transportmeasuring means to enhance its electrical sealing properties can bedetermined empirically (see examples). The ion transport measuring meanscan be placed in a solution of metal salt for any length of time,preferably for more than one minute, and more preferably for more thanabout five minutes. Salt solution treatment can be done under anynon-frozen and non-boiling temperature, preferably at greater or equalthan room temperature.

Where treatments such as baking, fire polishing, or laser polishing areemployed, they can be used to enhance the smoothness of a glass orsilica surface. Where laser polishing of a chip or substrate is used tomake the surface surrounding an ion transport measuring means moresmooth, it can be performed on the front side of the chip, that is, theside of the chip or substrate that will be contacted by a samplecomprising particles during the use of the ion transport measuring chipor device.

Appropriate temperatures and times for baking, and conditions for fireand laser polishing to achieve the desired smoothness for improvedsealing properties of ion transport measuring means can be determinedempirically.

In some aspects of the present invention, it can be preferred to rinsethe ion transport measuring means, such as in water (for example,deionized water) or a buffered solution after acid or base treatment, ortreatment with an oxidizing agent, and, preferably but optionally,before using the ion transport measuring means to performelectrophysiological measurements on membranes, cells, or portions ofcells. Where more than one type of treatment is performed on an iontransport measuring means, rinses can also be performed betweentreatments, for example, between treatment with an oxidizing agent andan acid, or between treatment with an acid and a base. An ion transportmeasuring means can be rinsed in water or an aqueous solution that has apH of between about 3.5 and about 10.5, and more preferably betweenabout 5 and about 9. Nonlimiting examples of suitable aqueous solutionsfor rinsing ion transport measuring means can include salt solutions(where salt solutions can range in concentration from the micromolarrange to 5M or more), biological buffer solutions, cell media, ordilutions or combinations thereof. Rinsing can be performed for anylength of time, for example from minutes to hours.

Some preferred methods of treating an ion transport measuring means toenhance its electrical sealing properties include one or more treatmentsthat make the surface more electronegative, such as treatment with abase, treatment with electron radiation, or treatment with plasma. Notintending to be limiting to any mechanism, base treatment can make aglass surface more electronegative. This phenomenon can be tested bymeasuring the degree of electro-osmosis of dyes in glass capillariesthat have or have not been treated with base. In such tests, increasingthe electronegativity of glass ion transport measuring means correlateswith enhanced electrical sealing by the base-treated ion transportmeasuring means. Base treatment can optionally be combined with one ormore other treatments, such as, for example, treatment with heat (suchas by baking or fire polishing) or laser treatment, or treatment withacid, or both. Optionally, one or more rinses in water, a buffer, or asalt solution can be performed before or after any of the treatments.

For example, after manufacture of a glass chip that comprises one ormore holes as ion transport measuring means, the chip can be baked, andsubsequently incubated in a base solution and then rinse in water or adilution of PBS. In another example, after manufacture of a glass chipthat comprises one or more holes as ion transport measuring means, thechip can optionally be baked, subsequently incubated in an acidsolution, rinsed in water, incubated in a base solution, and finallyrinsed in water or a dilution of PBS. In some preferred embodiments, thesurfaces of a chip surrounding ion transport measuring means can belaser polished prior to treating the chip with acid and base.

To facilitate batch treatment of glass biochips, we have used thetreatment fixtures illustrated in FIG. 14. FIG. 14A shows a single layertreatment fixture that can fit into a glass jar containing acid, base,or other chemical solutions. FIG. 14B shows the stacked treatmentfixture. The fixture is made of acid and base resistant materials suchas cyclo-olefin polymer, polyphenylene ether/PPO, NORYL®, ZEONOR®,polytetrafluoroethylene, TEFLON™, etc. Glass pins are inserted and heldin the holes, so that one piece of glass biochip to be treated is fitinto one slot between two glass pins. Multiple layers of these racks canbe stacked up to fit into one glass container. This design also allowsmechanisms of moving fluid to occur such as that brought about by arotary or reciprocal shaker or a magnetic stir bar.

In some aspects of the present invention, it can be preferable to storean ion transport measuring means that has been treated to have enhancedsealing capacity in an environment having decreased carbon dioxiderelative to the ambient environment. This can preserve the enhancedelectrical sealing properties of the ion transport measuring means. Suchan environment can be, for example, water, a salt solution (including abuffered salt solution), acetone, a vacuum, or in the presence of one ormore drying agents or desicants (for example, silica gel, CaCl₂ or NaOH)or under nitrogen or an inert gas. Where an ion transport measuringmeans or structure comprising an ion transport measuring means is storedin water or an aqueous solution, preferably the pH of the water orsolution is greater than 4, more preferably greater than about 6, andmore preferably yet greater than about 7. For example, an ion transportmeasuring means or a structure comprising an ion transport measuringmeans can be stored in a solution having a pH of approximately 8.

Glass chips that have been base treated and stored in water with neutralpH levels can maintain their enhanced sealability for as long as 10months or longer. In addition, patch clamp chips bonded to plasticcartridges via adhesives such as UV-acrylic or UV-epoxy glues can bestored in neutral pH water for months without affecting the sealingproperties.

We have also tested patch clamp biochips and cartridges that were storedin a dry environment with desiccant for 30 days. The chips werere-hydrated and tested for sealing. In one experiment, we got 6/7 sealsfor the dry-stored chips. Similarly, we stored mounted chips in dryenvironment and were able to obtain seals after a few weeks of storage.

Dehydration can, however, reduce the sealability of chemically treatedchips. To improve the seal rate for dry-stored chips, NaOH, NaCl, CaCl₂and other salt or basic solutions can be used to rejuvenate the chipsout of dry storage to restore the sealability.

The present invention also includes methods of shipping or transportingion transport measuring means modified by the methods of the presentinvention to have enhanced electric sealing properties and structurescomprising ion transport means that have been modified using the methodsof the present invention to have enhanced electric sealing properties.Such ion transport measuring means and structures comprising iontransport measuring means can be shipped or transported in closedcontainers that maintain the ion transport measuring means in conditionsof low CO₂ or air. For example, the ion transport measuring means can besubmerged in water, acetone, alcohol, buffered solutions, saltsolutions, or under nitrogen (N₂) or inert gases (e.g., argon). Wherethe ion transport measuring means or structure comprising an iontransport measuring means is stored in water or an aqueous solution,preferably the pH of the water or solution is greater than 4, morepreferably greater than about 6, and more preferably yet greater thanabout 7. For example, an ion transport measuring means or a structurecomprising an ion transport measuring means can be shipped in a solutionhaving a pH of approximately 8.

In one method of shipping a chip that has been treated to have enhancedsealing properties, the ion transport measuring devices comprisingbase-treated chips are shipped such that the chips are loaded up sidedown. The package for commercial shipments is designed to holdcartridges up side down, although the up side up configuration can alsobe used for shipping. To allow easy opening and facilitate automation insequential loading of the devices onto apparatuses for use, a blisterpack with film sealing is designed. As illustrated in the FIG. 15, ablister pack is provided in the form of a molded plastic frame with anopening on both top and bottom sides for film sealing. The sealing filmor “lidstock” is a thin foil with temperature activated adhesive and aninert coating such as EVA (ethyl vinyl acetate) polymer. For wet (water)storage, the blister pack is first sealed from top (the opening side,flipped over, and the cartridges are loaded up side up. A preservativesolution such as water is then injected into each well and the rest ofthe open space in each chamber of the package. Another lidstock film isthen used to seal the blister package from the bottom. The blisterpackage can be optionally sterilized with radiation for long shelf life.

Yet another aspect is related to the shipping of laser processed glasschips as finished goods between to production processes, particularly ifthe two processes are in different production locations. The currentinvention describes a shipping fixture allowing individual placement andsecuring of laser-processed glass chips for shipment. The samefixture-chips assembly is then directly used for subsequent chemicalprocessing. FIG. 16 is an illustration of the closed (left) and opened(right) shipping fixture. To withstand strong acid and base treatment,the shipping fixtures are molded with inert materials such as Noryl,Teflon, and Zeonor. A stack of these fixtures can be secured in onecontainer for chemical treatments, or for shipping in aqueous solutionssuch as water. The liquid shipping provides buffering for vibrationsduring transportation, giving maximum protection of glass chips frombeing damaged.

The present invention also includes ion transport measuring meanstreated to have enhanced electrical sealing properties, such as bymethods disclosed herein. The ion transport measuring means can be anyion transport measuring means, including those disclosed herein. Thepresent invention also includes chips, pipettes, substrates, andcartridges, including those disclosed herein, comprising ion transportmeasuring means treated using the methods of the present invention tohave enhanced electrical sealing properties.

The present invention also includes methods of using ion transportmeasuring means and structures comprising ion transport measuring means,such as biochips, to measure ion transport activity or functions of oneor more particles, such as cells. The methods include: contacting asample comprising at least one particle with an ion transport measuringmeans that has been modified to enhance the electrical seal of aparticle or membrane with the ion transport measuring means, engaging atleast one particle or at least one membrane on or at the modified iontransport measuring means, and measuring at least one ion transportfunction or property of the particle or membrane. The methods can bepractices using the methods and devises disclosed herein. Generally, themethods of the present invention provide the following characteristics,but not all such characteristics are required such that somecharacteristics can be removed and others optionally added: 1) theintroduction of particles into a chamber that includes a biochip of thepresent invention, 2) optionally positioning particles at or near an iontransport detection structure, 3) electronic sealing of the particlewith the ion transport detection structure, and 4) performing iontransport recording. Methods known in the art and disclosed herein canbe performed to measure ion transport functions and properties usingmodified ion transport measuring means of the present invention, such assurface-modified capillaries, pipette, and holes and apertures onbiochips and channel structures.

Methods for Measuring the Surface Energy of the Surface of a ChemicallyTreated Ion Tranport Measuring Biochip

Another aspect of the current invention originated from the need for aninexpensive, fast, and sensitive method to measure surface energy onsolid/liquid surface such as that of a chemically treated ion transportmeasurement biochip.

The method includes: dispensing a drop of defined volume of water or anaqueous solution on a surface, measuring the time it takes for the dropto evaporate; and estimating the relative or absolute surface energy ofthe surface based on the evaporation time, and the difference inevaporation time with respect to control samples.

The method can be used to determine the hydrophilicity of at least aportion of the surface of an ion transport measuring chip. In this case,a drop of water or aqueous solution is dispensed on the surface of abiochip comprising at least one ion transport measuring means,preferably a biochip that has been chemically treated to improve itselectrical sealing properties. Evaporation is monitored, and the timeelapsed between the time the drop contacts the chip and the time it hastotally evaporated is measured. The elapsed time is used as an index forhydrophilicity. This index can be used to determine whether a chemicallytreated chip is within the optimal range for achieving high resistanceelectrical seals.

Evaporation can be monitored by diffraction, reflectance, orinterference at the surface where the drop is deposited, or simply byvisual observation. Evaporation can also be monitored by measuring thechange in intensity or other physical or chemical properties of a dye ortracer agent that has been used to color or label the solution.

The invention uses the evaporation time of a liquid drop on a solidsurface as a measure of the solid/liquid surface energy. The method hasvery low cost (an accurate liquid dispenser is the only equipmentneeded). It is also very fast and accurate for low surface energysystems.

The contact angle of a liquid drop on a solid surface is a measure ofthe surface energy, assuming constant liquid/air surface energy. Verylow liquid/solid energy results in extremely small contact angles (closeto 0 degrees). For that reason, contact angle measurements might not bea very sensitive method for low surface energy systems.

When a liquid drop with fixed volume is in contact with a solid surface,the air/liquid surface of the drop will be inversely proportional to theliquid/solid surface energy. Lower liquid/solid surface energy willresult in bigger spreading of the drop. The evaporation of the drop willbe proportional to the air/liquid surface area at any given moment. Thusthe evaporation time will be proportional to the liquid/solid surfaceenergy.

Using the drop evaporation technique, we have demonstrated that theevaporation time of a 0.25 microliters water drop is 2.5 times shorterfor a highly hydrophilic glass surface (treated with base) compared tochemically untreated glass.

Methods of Manufacturing Chips for Ion Transport Measurement Devices

Yet another aspect of the present invention is a method of making a chipfor ion transport measurement devices by fabricating a chip thatcomprises multiple rows of ion transport measuring holes andsubsequently breaking the chip into discrete segments that comprise asubset of the total number of ion transport measuring holes.

In this method, a glass sheet is pre-processed with a laser to createpatch clamp recording apertures, and preferably treated chemically toimprove sealability as described in this application. The glass sheethas also been pre-scored with a laser to produce mark lines by whichsets of holes can be separated from one another. Preferably, the marklines are continuous slashes that go through the glass to a depth ofabout 50% or more of the thickness of the sheet.

In some preferred embodiments, an injection molded multi-unit well plateis bonded to the glass with adhesives so that each well of the plate isin register with one of the ion transport recording holes. Unitscomprising a portion of the multi-unit well plate and a portion of theglass chip are separated later by two metal plates closing in from twosides of the scored mark lines against the glass sheet, followed bybending of the bonded multi-well devices along with the metal plates andpulling of the segments away from each other (see FIG. 17).

This approach allows for low cost, automated assembly of single well orlow- density arrays, such as 16-well planar patch clamp consumables.This method of manufacture improves automation, and reduces individualunit assembly time.

High Density Ion Transport Measurement Chips

Another aspect of the present invention is a high density, highthroughput chip for ion transport measurement. A high density chip forion transport measurement comprises multiple ion transport measuringholes. The invention also encompasses methods of making high-densityconsumable patch clamp arrays for ultra high throughput screening of iontransport function.

A high density chip for ion transport measurement comprises at least 24ion transport measuring holes, preferably at least 48 ion transportmeasuring holes, and more preferably, at least 96 ion transportmeasuring holes. A high density, high throughput chip for ion transportmeasurement of the present invention can comprise at least 384 iontransport measuring holes, or at least 1536 ion transport measuringholes.

A high density ion transport measuring chip can be made using a silicon,glass, or silicon-on-insulator (SOI) wafer. The wafer is firstwet-etched to create wells on the top surface, and then laser drillingis used to form the through holes. The dimensions of the wafer and thewells can vary, however, in preferred embodiments in which a 1536 wellarray is fabricated, the thickness of the wafer can range from about 0.1micron to 10 millimeters, preferably from about 0.5 micron to 2millimeters, depending on the substrate.

For wafers in the range of 1 millimeter thick, the etching toleranceshould be within 2% if the through holes are approximately 30 microns indepth. This applies to Si wafers etched with alkaline solutions such asKOH or glass wafers etched with buffered HF. With SOI wafers, a definedthickness of SiO₂ covers the Si wafers, and etching of the wells throughthe Si side with KOH will stop at the SiO₂ interface. This way thethickness of the remaining material is consistent across the wholewafer, and even consistent among different batches of etched wafers.This permits laser drilling on these etched substrates to be morestandardized, and reduces the time needed for laser measurement. In apreferred embodiment, the etched Si wells have a volume of approximately2 microliters, assuming a footprint of approximately 2 millimeters×2millimeters for each well that extends as a prism or inverted pyramidshape through the Si substrate during anisotropic etching, leaving adistance of approximately 1 millimeter between adjacent wells.

In one design, the bottom of the chip can be sealed against a singlecommon reservoir for measuring solution that is connected to a commonreference electrode, while individual recording electrodes can beconnected at the upper surface directly or via electrolyte bridges.

Alternatively, a structure with 1536 or any preferred number ofindividual isolated chambers can be sealed against the bottom of the1536 or any preferred number of well plate so that each chamber is inregister with a well. In some designs of this embodiment, the topsurface of the SOI wafer can be a common electrode, with theconductivity of Si material being adequate to provide electricalconnection; however, additional metal coating on the top surface(applied before etching as mask layer) can increase conductivity of theupper surface. Wet etching that creates the wells removes this metalcoating from the wells themselves. Chemical treatment with acid and/orbase can optionally be performed on the chip for improved sealing.

Another way to make a high density chip is to use very thin wafers madeof glass, SiO₂, quartz, Si, PDMS, plastics, polymers, or othermaterials, or a thin sheet, with thickness between about 1 micron andabout 1 millimeter. Laser drilling can be performed on such sheets tocreate through holes. A separate, “well plate” with 1536 or anypreferred number of wells, manufactured by molding, etching,micro-machining or other processes, is then sealed against the holes viagluing or by using other bonding methods.

FIG. 18 shows a high density array made on a Si, glass, or SOI wafer. Itis made with a wet etch process, which creates the wells on the topsurface, followed by laser drilling through the remaining of thematerial on the bottom of each of the wells (FIG. 19). The laserdrilling of the holes can be from the front or back side of the chip.

For high density ion transport measuring chips, either a “standard” orinverted drilling configuration can be used as described herein.

Methods for Assembling Ion Transport Measurement Cartridges

Use of Adhesives

An ion transport measurement cartridge comprises one or more upperchamber pieces bonded via adhesive or other means to one or more iontransport measurement chips that have been treated to have enhancedelectrical sealing properties which contain at least one microfabricatedion transport measurement aperture (hole), optionally but preferablydrilled by a laser. The one or more ion transport measurement chips areoptionally laser polished on the side of the small exit hole, andtreated with a combination of acid and base treatment as describedherein.

The present invention also includes a method of assembling ion transportmeasurement cartridges by bonding the ion transport measurement chip(s)with an upper chamber piece. In one embodiment, an ion transportmeasurement chip containing one or more ion transport measuringapertures is bonded to an upper chamber piece via a UV-activatedadhesive, such that each well of the upper chamber piece is in registerwith a recording aperture on the ion transport measurement chip, and thesmaller, exit holes from laser drilling of the ion transport measuringholes are exposed to the wells of the upper chamber piece.

To facilitate efficient assembly, a registration bump can preferably bemolded on the bottom of the upper chamber piece so that when the biochipis pressed against the bump and shoulder at the bottom of the upperchamber piece, the recording apertures on the ion channel measurementchip are in register with the wells of the upper chamber piece.

Preferred UV adhesive include, but are not limited to, UV-epoxy,UV-acrylic, UV-silicone, and UV-PDMS.

The UV dose required to completely cure the UV adhesive can at timesinactivate the treated surface of the chip. To avoid UV radiation tochip surface areas near the recording apertures where seals are tooccur, a mask made of UV-permeate glass on which spots of size between0.5 to 5 mm are provided by depositing a thin metal layer or paint(preferably a dark or black) layer. One such mask that can be mounted tothe cartridge being assembled is illustrated in FIGS. 20, 21, and 22.

Pressure Mounting

As an alternative to glue-based bonding, the upper chamber piece can bedesigned to allow an O-ring type of gasket made with PDMS to be used asseal cushion between the upper chamber piece and a biochip during asandwich-type pressure mounting procedure (see FIG. 23).

Biochip Device for Ion Transport Measurement Comprising Fluidic ChannelChambers

A further aspect of the present invention is a flow-through fluidicchannel ion transport measuring device that can be part of a fullyautomated ion transport measuring device and apparatus. This devicecomprises a planar chip that comprises ion transport measuring holes,and upper and lower chambers on either side of the chip that are fluidicchannels. One or more fluidic channels is positioned above the chip andone or more fluid channels is positioned below the chip. Apertures arepositioned in the fluidic channels such that an ion transport measuringhole in the chip has access to an upper fluidic channel (serving as anupper chamber) and a lower fluidic channel (serving as a lower chamber).

A chip of a fluidic channel ion transport measuring device can havemultiple ion transport measuring holes, and each of the holes can be influid communication with an upper fluidic channel and a lower fluidicchannel. The upper fluidic channels can be connected with one another,when the lower fluidic channels are independent; or the upper fluidicchannels can be independent while the lower fluidic channels can beconnected with one another. In a yet another alternative, upper fluidicchannels that service different ion transport measuring holes can beseparate from one another and the lower fluidic channels that servicedifferent ion transport measuring holes can also be separate from oneanother.

In FIG. 24, a dashed line outlines the planar patch clamping chip, andupper and lower fluidic channels for an extracellular solution (ES) andintracellular solution (IS), are shown. The upper and lower channels areinterfaced at a point where the recording aperture of the planarelectrode resides. Separate fluidic pumps drive the flow of fluidsthrough the two (upper and lower) fluidic channels. Recording andreference electrodes external to the fluidic patch clamp chip areconnected via a electrolyte solution bridge to the top and bottomfluidic channels. A pressure source such as a pump with pressurecontroller that can generate both positive and negative pressures islinked to the lower fluidic channels. A multi-way valve is used toconnect the lower fluidic channel to different solution reservoirs (IS1,IS2, etc), while a multi-way valve is used to connect the upper fluidicchannel to cell reservoirs, compound plate, wash buffers and othersolutions.

This design has several advantages. The external electrodes can be ofmultiple use, but replaceable. This reduces the cost of the biochip. Theflow-through fluidics of both the upper and lower chambers minimizes thegeneration of air bubbles. Importantly, the closed fluidic channelsallow for controlled delivery of low volume fluids without evaporation.

Methods of Preparing Cells for Ion Tranport Measurement

In a further aspect of the present invention, methods for isolatingattached cells for planar patch clamp electrophysiology are provided.Conventional cell isolation methods by non-enzymatic, trypsin, orreagent-based methods will not produce cells that are in optimalcondition for high throughput electrophysiology. Typically cellsproduced by available protocols are either over-digested and tend tofunction less than optimally in planar patch clamp studies, orunder-digested and resulting in cell clumps with the cell suspension. Inaddition, the cells isolated by conventional methods tend to have largeamounts of debris which are a major source of contamination at therecording aperture. The current protocols are optimized for better cellhealth, single cell suspension, less debris and good patch clampperformance. The current protocols can be used to isolate cells for anypurpose, particularly when cells in an optimal state of health andintegrity are desirable, including purposes that are not related toelectrophysiology studies.

This invention was developed to produce suspension CHO and HEK cellsthat give high quality patch clamp recording when used with chips anddevices of the present invention. Parameters such as cell health, sealrate, Rm (membrane resistance), Ra (access resistance), stable wholecell access, and current density, were among the parameters optimized.The method includes: providing a population of attached cells, releasingthe attached cells using a divalent cation solution, anenzyme-containing solution, or a combination thereof; washing the cellswith a buffered cell-compatible salt solution; and filtering the cellsto produce suspension cells that give high quality patch clamprecordings using ion transport measuring chips.

Enzyme-Free Cell Preparation

Enzyme-free dissociation is desirable when an ion transport expressed ona cell surface can be digested by enzymatic methods, thereby causing achange in ion transport properties. Enzyme-free methods involve adissociation buffer that is either Ca⁺⁺-chelator-based ornon-Ca⁺⁺-chelator-based. The former is typically a solution of EDTA,while the latter can be calcium-free PBS. In such methods, attachedcells grown on plates are first washed with calcium-free PBS, and thenincubated with the dissociation buffer. In case of the calciumchelator-based dissociation, the dissociated cells must be washed atleast once with a chelator-free solution before they can be used for iontransport measurement assays. The suspended cells are then passedthrough a filter, such as a filter having a pore size of from about 15to 30 microns (this can vary depending on the type of cells and theiraverage size).

Preparation of Cells using Enzyme

In some methods (see Example 6), trypsin is used to dissociate attachedcells. In such methods, the cells are typically rinsed with a solutiondevoid of divalent cations, and then briefly treated with trypsin. Thetrypsin digestion is stopped with a quench medium carefully designed toachieve the optimal divalent cation mix and concentration. In themethods provided herein, the suspended cells are then passed through afilter, such as a filter having a pore size of from about 15 to 30microns (this can vary depending on the type of cells and their averagesize).

Another enzyme-based method uses a preparation commercially availablefrom Innovative Cell Technology (San Diego). Accumax is an enzyme mixcontaining protease, collagenase, and DNAse. Example 6 provides aprotocol for CHO cells using Accumax and filtration.

Some preferred methods of the present invention use a combination ofenzyme-free dissociation buffer, Accumax reagent, and filtration toisolate high quality cells for patch clamping (see Example 6).

Pressure Profile Protocol for Ion Transport Measurement

The present invention also provides a pressure protocol control programlogic that can be used by an apparatus for ion transport measurement toachieve a high-resistance electrical seal between a cell or particle andan ion transport measuring means on a chip of the present invention in afully automated fashion. In this aspect, the program interfaces with amachine that can receive input from an apparatus and direct theapparatus to perform certain functions.

Typically it has required months to years of experience on the part ofan experimenter to master the techniques required to achieve andmaintain high quality seals during their experiments. It is an object ofthe invention to produce a pressure protocol for achieving andmaintaining seal quality parameters for automated patch clamp systems.The present invention provides a logic that can direct mechanical andautomated patch clamp sealing of particles and membranes.

The program logic includes: a protocol for providing feedback control ofpressure applied to an ion transport measuring means of an ion transportmeasuring apparatus, comprising: steps that direct the production ofpositive pressure; steps that direct the production of negativepressure; steps that direct the sensing of pressure; and steps thatdirect the application of negative pressure in response to sensedpressure in the form of multiple multi-layer if-then and loop logic, inwhich the positive and negative pressure produced is generated throughtubing that is in fluid communication with an ion transport measuringmeans of an apparatus, and in which negative pressure is sensed throughtubing that is in fluid communication with an ion transport measuringmeans of an apparatus. Preferably, these steps are performed in adefined order that depends on the feedback the apparatus receives. Thus,the order of steps of the protocol can vary according to a definedscript depending on whether a seal between a particle and the iontransport measuring means is achieved during the operation of theprogram, and the properties of the seal achieved.

An apparatus for ion transport measurement that is controlled at leastin part by the pressure program preferably comprises: at least one iontransport measurement device comprising two or more ion transport units(each comprising at least a portion of a biochip that has an iontransport measuring means, at least a portion of an upper chamber, andat least a portion of a lower chamber, and is in electrical contact withat least one recording electrode and at least one reference electrode),tubing that connects to the device and is in fluid communication withthe two or more ion transport measuring means of an apparatus, and pumpsor other means for producing pressure through the tubing. Preferably,the apparatus is fully automated, and comprises means for deliveringcells to upper chambers (such means can comprise tubing, syringe-typeinjection pumps, fluid transfer devices such as one or more automatedfluid dispensers) and means for delivering solutions to lower chambers(such means can comprise tubing, syringe-type injection pumps).

Preferably, in addition to promoting and maintaining a high resistanceseal, the pressure protocol program can also direct the rupture of acell or membrane delineated particle that is sealed to an ion transportmeasuring means. Such rupture can be by the application of pressureafter sealing, and can be used to achieve whole cell access.

In operation, the program directs the apparatus to generate a positivepressure in the range of 50 Torr to 2000 Torr, preferably between 500and 1000 Torr, to purge any blockage of the recording holes. Then theprogram directs the apparatus to generate a positive holding pressurebetween 0.1 to 50 Torr, preferably between 1 to 20 Torr to keep therecording aperture of an ion transport measuring chip clear of debrisduring the addition of cells to the upper chamber. After cell addition,the program directs the release of pressure and holds the pressure atnull long enough to allow cells to approximate the aperture. The programthen directs a negative pressure to be applied draw a cell onto (andpartly into) the ion transport recording aperture for landing and theformation of a gigaohm seal. Additional pressure steps as describedExample 7 may be required for achieving gigaohm seals if a seal does notoccur upon cell landing.

To achieve whole-cell access, negative pressure is increased inprogressive steps until the electrical parameters indicate theachievement of whole-cell access. Alternatively, the program can directthe application of a negative pressure to a “sealed” cell that isinsufficient to gain whole-cell access, and then use a electric “zap”method to disrupt the membrane patch within the aperture and therebyachieve whole-cell access. Upon achieving whole-cell access the pressureis released either immediately, or held for a few seconds then released,depending on the cell quality. Finally, during whole-cell accessprocedures, the seal quality could be improved after access has beenachieved, then held at optimal parameters by a more complex pressureprotocol.

The pressure protocol involves many branch-points or “decisions” basedupon feedback from the seal parameters. It is easiest to describe theprotocol as a series of steps in programming logic. An example of suchlogic (underlined items are user-defined parameters) is provided asExample 7.

Novel Ion Transport Measuring Biochip Designs

The present invention also includes novel methods of making high densityand/or multiplex ion transport measuring biochips and biochips made bythese methods. These devices can be used to record ion transportactivity of more than one particle or cell simultaneously or in rapidsequence. In preferred aspects, the ion transport measuring biochips andbiochips made by these methods are designed to be high density the iontransport measuring biochips. By “high density” is meant that the chipscomprise a large number of ion transport measuring means. Typically, theion transport measuring means are holes through the surface of thebiochip, and a high density transport measuring biochip has multiple iontransport recording sites via multiple holes. In this way, multipleassays can be conducted simultaneously, or in rapid sequence, allowingfor high-throughput ion transport measuring assays that can facilitate,for example, compound assays.

As used herein, “high throughput” means high quantity of independentdata collected in a defined period of time. For example, 48 or moreassays that can be conducted within a short time span where multipleassays are initiated simultaneously or in rapid succession, then shareexperimental time as parallel or multiplexed recordings, and tencompleted simultaneously or independently but in parallel (less than onehour from loading of cells to completing ion channel recording,preferably, less than one half hour from loading of cells to completingion channel recording, and more preferably, less than fifteen minutesfrom loading of cells to completing ion channel recording). Morepreferably, more than 96 high throughput ion transport measurements canbe completed in less than one half hour, and more preferably yet, thehigh density ion transport measuring devices of the present inventionare capable of performing more than 100 ion transport assays within onehalf hour or less. In some preferred aspects of these embodiments, highdensity ion transport measuring devices can perform hundreds or over onethousand assays within one half hour or less. For example, in somepreferred aspects of high density ion transport measuring devicesdescribed herein, the devices can be designed to perform 384 assays or,for example, 1536 assays, within one half hour or less. For anotherexample, 48 or more assays that can be conducted within a time spanduring which continuous and repetitive data sampling are performed forkinetic studies with high temporal resolution. In another example,multiple lower density assays, such as 16-assay devices, may be utilizedin parallel to result in a high density assay.

While the devices herein can be described as high-throughput, thedesigns are not limited to high throughput uses and can be used for anynumber of ion transport assays, in assays that can last from seconds toseveral hours.

MCP-Based Chip

One aspect of the present invention is an ion transport measuring devicethat comprises a microchannel plate (MCP). Microchannel glass platesthat comprise an array of microchannels and their fabrication are knownin the art of electronics and optics for their use as electronmultipliers and photomultipliers. Some aspects of their fabrication anduse are described in Wiza (1979) Microchannel Plate Detectors NuclearInstruments and Methods 162: 587-601. In brief, they can be made byproviding glass fibers that have a core glass and a cladding thatcomprises lead glass. The fibers are arranged together side-by-side in adesirable configuration, drawn, surrounded by a glass envelope, andfused to produce a boule. The boule can be sliced (cutting perpendicularto the fiber lengths) to produce slices that are cross-sections of theboules. These slices can be finished, for example, by polishing. Thecores of the glass fibers are then chemically etched away, to form themicrochannel plate.

An MCP made for use as part of an ion transport measuring device can bemade by fusing from 2 to over 1,000 glass fibers. An MCP ion transportmeasuring chip can, for example, be a high-density ion transportmeasuring chip that comprises 48 or more microchannels that serve as iontransport measuring holes, and preferably, 96 or more microchannels thatserve as ion transport measuring holes. The core of the fibers (theportion made of etchable glass) used to make an MCP chip can be as wideas 40 microns in diameter (for chips used for ion transport assays usinglarge cells, such as oocytes) but preferably are from 0.2 to 8 micronsin diameter, and are more preferably from 0.5 to 5 microns in diameter,even more preferably from 0.5 to 3 microns in diameter, and mostpreferably about 2 microns in diameter. The thickness of the lead glasscladding around the core can vary depending on the desired spacing ofthe resulting ion transport measuring holes. The length of the fibersused in making an MCP ion transport measuring chip are not limiting, andcan be of any feasible length. Preferably, after fusing the glassfibers, the boule is sliced into sections that are from about 5 micronsto 5000 microns thick, most preferably from about 10 to about 50 micronsthick.

The core glass fibers can be randomly arranged or configured into apattern to make the boule.

In one design, depicted in FIG. 35, the area surrounding the iontransport measuring holes on the upper side of the MCP chip can bechemically wet-etched to produce microwells that can be used as upperchambers. These upper chambers can be used for measuring solution, cellsor particles, and test compounds. The MCP chip can be bonded to a bottompiece that comprises one or more lower chambers. The MCP plate can alsobe bonded to an upper piece that comprises the ES chambers.

The surface of the MCP chip can be chemically treated, such as usingmethods disclosed herein, to enhance the electrical seal of a particleor membrane with the ion transport measuring means. The entire MCP chipor a portion thereof can be treated to enhance its electrical sealingproperties. Preferably, at least a portion of the surface of the MCPchip to which cells or particles are to be sealed is treated. One orboth surfaces, or one or more portions of one or both surfaces, of theMCP chip can also be coated, or one or more portions of one or bothsurfaces, with one or more materials that can increase its sealingproperties. In some embodiments, one or both surfaces, or one or moreportions of one or both surfaces, of the MCP chip can be coated with oneor more hydrophobic materials that can be used to promote fluidicisolation of individual microwells of the MCP chip. Designs in whichhydrophobic surfaces are used to promote fluidic isolation of individualmicrowells of a chip are further described as Aspect 19 of thisapplication (below).

In designs in which the bottom piece forms individual lower chambers,reference electrodes can be within or electrically connected with theupper wells and recording electrodes can be within or electricallyconnected with the lower wells for ion transport measurement, orrecording electrodes can be within or electrically connected with theupper wells and reference electrodes can be within or electricallyconnected with the lower wells for ion transport measurement.

In one possible design involving etched microwells on the upper surface,a common reference electrode can connect all of the upper microwells.The electrode, which can be a conductive material such as metal, canfollow paths along the top surface of the MCP chip and contact measuringsolution only where it contacts the interior of the microwells. Theelectrode can optionally be coated with a nonconductive material whereit traverses the chip surface, and be exposed where it contacts theinterior of the wells.

Where a device comprising an MCP chip is configured to have a commonelectrode that contacts multiple lower wells of the device, the samedesign can be used.

In designs in which the bottom piece forms a bottom chamber thatcontacts more than one ion transport measuring hole, the bottom chamberpreferably comprises or is in electrical contact with a referenceelectrode, and individual upper chambers comprise individual recordingelectrodes. Alternatively, the bottom piece can comprise multiple lowerchambers with individual recording electrodes, and the device has acommon upper chamber with a reference electrode. In this embodiment,compounds can be added using compound delivery mechanisms such as, forexample, fluid block delivery, chamber separators, or other mechanismsdescribed herein.

In an alternative design for an ion transport measuring device thatcomprises an MCP chip, upper chambers can be constructed by attaching amanufactured piece that comprises well openings such that each well ofthe upper chamber piece aligns with one of the ion transport measuringholes (the microchannels of the MCP). Individual upper chamberspreferably have a volume of from about 0.5 microliters to about 5milliliters, and more preferably from about 2 microliters and about 2milliliters, and more preferably yet between about 10 microliters andabout 0.5 milliliter. The upper chamber piece can be irreversibly orreversibly attached to the MCP ion transport measuring chip usinggaskets, clamps, adhesives, welding, or other means. The upper chamberpiece can comprise glass, ceramics, coated metals, or (preferably)plastics or polymers. In one preferred embodiment, the upper chamberpiece comprises a separate MCP. In this design, the glass fibers used tomake the upper chamber piece MCP are of a wider diameter than those usedto make the ion transport measuring chip MCP. The glass fibers used tomake the upper chamber piece MCP also comprise a cladding of sufficientthickness to provide chamber spacing over the ion transport measuringholes of the ion transport measuring chip MCP. Conduits can connect tothe wells of the upper chamber piece for the addition of solutions,cells, or compounds. Alternatively, a fluid dispensing device caninterface with the upper chamber wells to dispense solutions, cells, orcompounds.

A lower chamber piece can also comprise multiple chambers that connectto individual ion transport holes of the MCP chip. The lower chamberpiece can be constructed by attaching a manufactured piece thatcomprises wells spaced such that each well of the lower chamber piecealigns with one of the ion transport measuring holes (the microchannelsof the MCP). The lower chamber piece can be irreversibly or reversiblyattached to the MCP ion transport measuring chip using gaskets, clamps,adhesives, welding, or other means. The upper chamber piece can compriseglass, ceramics, coated metals, or (preferably) plastics or polymers. Inone embodiment, the lower chamber piece comprises a separate MCP. Inthis design, the glass fibers used to make the upper chamber piece MCPare of a wider diameter than those used to make the ion transportmeasuring chip MCP. The glass fibers used to make the lower chamberpiece MCP also comprise a cladding of sufficient thickness to providechamber spacing over the ion transport measuring holes of the iontransport measuring chip MCP. Conduits can connect to the wells of thelower chamber piece for the addition of solutions, and allowingpneumatic control.

In using devices having individual upper chambers and individual lowerchambers recording electrodes (or connections to recording electrodes)can be provided in or attached to upper chambers, and referenceelectrodes (or connections to reference electrodes) can be provided inor attached to lower chambers. In the alternative, recording electrodes(or connections to recording electrodes) can be provided in or attachedto lower chambers, and reference electrodes (or connections to referenceelectrodes) can be provided in or attached to upper chambers.

In some preferred embodiments, however, a device that comprises an MCPion transport measuring chip can have a single lower chamber thataccesses all ion transport measuring holes of the MCP chip. In thiscase, the lower chamber can also comprise ceramics, coated metals,glass, plastics, or polymers, and preferably connects to conduits thatconnect to pressure sources and can deliver and remove fluids to andfrom the chamber. Pressure control may be performed from either bottomchambers or upper chambers, or both. In these embodiments, the lowerchamber preferably comprises or is in electrical connection with areference electrode during use of the device, and each upper chambercomprises or is in electrical connection with a recording electrodeduring use of the device.

In some other preferred embodiments, a device that comprises an MCP iontransport measuring chip can have a single upper chamber that accessesall ion transport measuring holes of the MCP chip. In this case, theupper chamber can also comprise ceramics, coated metals, glass,plastics, or polymers and it preferably comprises or is in electricalconnection with a reference electrode during use of the devices. In thiscase each lower chamber comprises or is in electrical connection with arecording electrode during use of the device. Pressure control may beperformed from either bottom chambers or upper chambers or both.

The present invention comprises ion transport measuring devicescomprising an MCP chip having greater than two through holes, and atleast one upper chamber. Preferably an ion transport measuring devicecomprising an MCP chip has multiple upper chambers that are reversiblyor irreversibly attached to the MCP chip. Preferably an ion transportmeasuring device that comprises an MCP chip can be reversibly orirreversibly attached to at least one lower chamber. The presentinvention also comprises ion transport measuring devices comprising anMCP chip having multiple microchannel through holes, and an MCP chiphaving multiple microchannel upper chambers.

The present invention also comprises methods of using MCP chips formeasuring ion transport activity and properties, as well as for otherassays.

Chip with Hydrophobic Surface Coating

Another aspect of the present invention is a hydrophobic ion transportmeasuring biochip that comprises ion transport measuring means in theform of holes having counterbores, where the counterbores are microwellupper chambers, and where the surface of the biochip has a hydrophobicsurface.

A hydrophobic ion transport measuring biochip of the present inventioncan have any number of holes, from 1 to more than one thousand. Inpreferred embodiments, a hydrophobic ion transport measuring biochip ishigh-density, and can be used for high throughput screening (such as,but not limited to, compound screening), and has 384 or more iontransport measuring holes. In some embodiments, a hydrophobic iontransport measuring biochip can have 1536 or more ion transportmeasuring holes.

A hydrophobic chip having microwell upper chambers can be made byproviding a suitable substrate, such but not limited to a glass, quartz,silicon, silicon dioxide, or one or more polymers, and coating thesubstrate with a hydrophobic material. Suitable materials for providinga hydrophobic coating include plastics and polymers, such as, forexample, polyethylene, polyacrylate, polypropylene, polystyrene, orpolysiloxane. After coating the chip, two or more holes are made, suchas by laser drilling into the chip. The laser drilling has the effect ofmelting and burning the polymer in the area surrounding the drilledhole, provided an uncoated (hydrophilic) surface in the area where acell (or other particle) can seal. Preferably, a counterbore is alsodrilled into the chip, where the counterbore can serve as a microwell onthe upper surface of the chip.

This design provides upper microwells (made by laser drilling) that arein liquid fluid isolation from one another, as the hydrophobic surfacebetween wells repels aqueous liquids such as buffers and measuringsolutions. The ion transport holes and areas immediately surroundingthem (such as counterbore microwells) have hydrophilic surfaces thathave been exposed by the laser drilling and therefore will retainbuffers and solutions. The upper microwells can be connected to a commonreference electrode that is coated with a nonconducting (andhydrophobic) material, such as a plastic or polymer used to coat thechip surface and traverses the surfaces of the chip. The electrode canbe uncoated where it contacts the microwells, so that the microwells arein electric communication without the possibility of solution exchangeor mixing between wells (see FIG. 36).

One preferred embodiment of the electrode on the hydrophobic chip is ametallized layer on the substrate coated by deposition, growing,condensing, or other means. The metallized layer can be removed at andnear the recording sites by laser shots or masking. The hydrophobiclayer is then coated on top of the metallized layer to allow for fluidicliquid separation between two adjacent recoding sites, leaving a ring ofmetallized layer uncovered near the recording sites to allow forelectrical connection of each recording sites (in form of a hole, or ahole and a microwell) with the metallized layer which services as areference electrode. The metallized layer can be made of any conductivematerial or materials including metals, non-metals, metal derivatives,or combinations thereof.

As alternatives, individual recording electrodes can also be physicallyor electrically connected (such as through electrolyte bridges) to eachof the upper chamber microwells. In these designs, there can beindividual or common lower chambers that engage the chip, and the one ormore lower chambers comprise or are electrically connected to one ormore reference electrodes.

Where the coating material is resistant to treatment chemicals, such asbase and/or acid, the surface of the hole on the hydrophobic chip can bechemically treated, such as by using methods described herein, toenhance the electrical sealing properties of the chip.

The present invention also includes methods of making the hydrophobicchip described herein, devices comprising the hydrophobic chip describedherein, where the devices can employ any feasible lower chamber,electrode, fluidic and pneumatic designs. The present invention alsoincludes methods of using a hydrophobic ion channel measuring chip tomeasure ion channel activity or properties of one or more cells orparticles.

Flexible Ion Transport Measurement (ITM) Chip

Another aspect of the present invention is a method of making a flexibleion transport measuring biochip that comprises a flexible sheet ofmaterial, preferably coated with glass, comprising multiple iontransport measuring holes. The flexible sheet of material can be woundaround a spool and unwound to form either a curved or an essentiallyflat surface for ion transport measurement. Alternatively, the flexiblesheet of material can be curved to form a tube, on the surface of whichion transport measurement assays can be performed.

The method comprises: providing a substrate that comprises a sheet offlexible material; creating (for example, by laser drilling, chemicaletching, micromachining, molding, etc) at least two holes in thesubstrate that extend through the substrate; and optionally coating thesubstrate with SiO₂ or glass to provide an ITM chip.

The substrate can comprise any material that can be provided as a thinsheet (for example, of within the range of between 5 and 5000 microns inthickness) and has a flexibility that allows the sheet to be curvedcompletely around (such as to make a tube) yet is hard and rigid enoughto allow manufacture of ion transport measuring holes through thesubstrate (that is, holes of a diameter within the range of from about0.2 to about 8 microns in diameter, although larger diameters can beused depending on the cell type to be assayed). For example, rubber,plastics, polymers or other flexible sheet materials can be used. Onesuch material is polyimide or Kapton. Kapton sheets of from about 5 to5000 microns in thickness, preferably from about 10 to about 200 micronsin thickness, can be laser drilled to produce through holes of withinthe range of from about about 0.2 to about 8 microns in diameter,preferably from about 0.5 to 5 microns in diameter, and more preferablyfrom about 0.5 to about 3 microns in diameter. Counterbores that can beused as microwells can also optionally be drilled into the polyimidesheet, as described herein. From 2 to over 50,000,000 holes can bedrilled into a single polyimide sheet, depending on the application,which can be further rolled around a spool. For example, where aflexible biochip is to be used as a “chip roll” in which section of theflexible biochip are used to be used sequentially, the sheet cancomprise a very large number of holes, a subset of which are to be usedin any given assay.

Before or after laser drilling of holes in the flexible substrate, thesubstrate is preferably treated or coated with a material that allowsfor efficient and high-resistance sealing of particles such as cells tothe ion transport measuring holes. The treatment or coating can compriseany material that promotes high-resistance sealing of particles such ascells to the ion transport measuring holes of the chip, and can compriseorganic or inorganic molecules, synthetic molecules (for example,polymers) or naturally occurring ones, in liquid or non-liquid form. Thecoated surface can be hydrophobic or hydrophilic, charged or uncharged,and can be linked to the substrate covalently or non-covalently. In onepreferred embodiment, the substrate is coated with glass.

If the coating is a naturally rigid material, such as glass, the coatingshould be thin enough, or physio-chemically altered to permit curving ofthe coated flexible sheet. The coating thickness can range from a singlemolecule layer to several micrometer. The optimal thickness for thedegree of curvature that is desirable (depending on the application) canbe determined empirically. The degree of curvature required in the useof the device that comprises the flexible biochip can also be adjusted(for example, by adjusting spool diameter, if the substrate is to bewound around a spool, or by adjusting tube diameter, if the substrate isto form a tube structure) to accommodate the coating if necessary.

The coating can be applied in any appropriate way: vapor deposition,dipping, soaking, direct application, spraying, “painting”, chemicalgrafting etc. If the coating is a polymer, in some cases polymerizationcan be promoted on the substrate surface. The coating can be adhered tothe substrate by absorption or chemical bonding. A glass coating can beapplied, for example, by vapor deposition (if the substrate material isresistant to the heat required, or by allowing solgel (hydrolyzedsiloxane) to polymerize to glass as it dehydrates on the substratesurface.

The surface of the flexible chip or portions thereof can optionally bechemically treated, such as by using the methods described herein, toimprove the electrical sealing properties of the chip.

In one aspect of this embodiment of the present invention, an iontransport measuring device can be made using a flexible ion transportmeasuring biochip of the present invention that is wound around a spool(see FIG. 37). In this embodiment, the leading edge of the flexiblebiochip extends from the spool to either a second spool, or to a guideinto which is inserted. The second spool or guide is positioned at aparticular distance from the first spool such that an expanse of theflexible biochip is extended to be used for ion transport assays. Theextended portion of the flexible biochip can be essentially flat orsomewhat curved. Preferably, the extended portion of the flexiblebiochip comprises multiple ion transport measuring holes that matchesthe number of wells in multi-well plate for compound testing.Preferably, the extended portion of the flexible biochip comprises atleast 12 ion transport measuring holes, more preferably, at least 24 iontransport measuring holes, even more preferably, at least 48 iontransport measuring holes, and yet more preferably, at least 96 iontransport measuring holes. For example, the extended portion of theflexible biochip can comprise 384 or 1536 ion transport measuring holes.

The present invention includes flexible ion transport measuring biochipsmade using these methods, and devices that include flexible iontransport measuring biochips.

An upper chamber piece can engage the upper side of the flexible biochipand a lower chamber piece can engage the lower side of the flexiblebiochip. In preferred aspects of these embodiments, the upper and lowerchamber pieces are reusable, and the flexible biochip is single-use. Inthese aspects, the upper and lower chamber pieces reversibly engage theflexible biochip for ion transport assays. Upon completion of a set ofassays, the upper and lower chamber pieces disengage and move away fromthe flexible biochip, a new section of the flexible biochip is unwoundfrom the spool as the leading edge is pulled through guides and the oldportion is optionally wound on a second spool, similar to camera filmwinding (in an alternative the used section can be pulled through guidesand clipped off, similar to use of a tape dispenser). The new section ofthe flexible biochip that is unwound from the spool is to be used in thesubsequent assay. The upper chamber piece and lower chamber piece(preferably one or both is reusable, but this is not a requirement ofthe present invention) now move to engage the new extended portion ofthe flexible biochip.

In aspects in which the extended portion of the flexible biochip issomewhat curved, such as by curving against the surface of another,“chamber spool” (FIG. 37B), in which the contact surface of the spoolalso comprises the upper or lower chamber pieces, the upper and lowerchamber pieces can be adapted to fit a curved biochip.

The upper chamber piece, the lower chamber piece, or both can be part ofa chamber “wheel” in which multiple chamber pieces, each of which isused in performing a set of assays, can sequentially engage the flexiblebiochip. For example, a first set of assays can be performed using thefirst extended portion of the flexible biochip and a first lower chamberpiece that is part of a chamber wheel that can rotate below the surfaceof the flexible biochip. Upon completion of the first set of assays, theused portion of the flexible biochip is pulled away from the wheel as anew portion of flexible biochip comes into proximity with the chamberwheel. During this period of time, the chamber wheel rotates so that theused chamber piece moves away from the assay site, and a new chamberpiece also attached to the wheel engages the new extended portion offlexible biochip at the assay site.

Various upper and lower chamber configurations can be combined with theflexible biochip. For example, an upper chamber piece that engages theflexible biochip can have multiple upper chambers, such that each iontransport measuring hole is associated with a single upper chamber, anda lower chamber piece can also have multiple lower chambers, such thateach ion transport measuring hole is associated with a single lowerchamber. It is also possible to have a single lower chamber thataccesses all of the ion transport holes used in an assay and multipleindividual upper chambers. In other cases a single upper chamber thataccesses all of the ion transport holes used in an assay and multipleindividual lower chambers. Different chamber arrangements can havedifferent electrode connections and connections to fluidic and pneumaticchannels.

In one preferred design, both the upper chamber piece and the lowerchamber piece comprise multiple chambers that align with the extendedportion of the flexible biochip such that each ion transport measuringhole is associated with a single upper chamber and a single lowerchamber. In this design, cells, extracellular solutions, and compoundscan be added to the top chambers either by individual conduits or byfluid dispensing systems. Pneumatic conduits connect with the lowerchambers to produce high resistance seals. Electrodes can be provided inthe reusable chamber pieces, or can be provided in fluid conduits or aspart of the ion transport recording machinery that can be brought intoelectrical contact with the chambers through electrolyte bridges.

In yet another aspect of a flexible ion transport measurement biochip,the flexible biochip can form an at least partially tubular structure.The flexible biochip can form at least a portion of a tube. Where theflexible biochip does not form the complete circumference of a tube, thesame flexible substrate material or a different material can form theremainder of the circumference of the tube. Where the flexible biochipdoes not form the complete circumference of a tube, the same flexiblesubstrate material or a different material can form a basin or bottomsurface of a trough-like structure that is continuous with the curvedchip but can be at least in part flat or have a lesser degree ofcurvature. In this embodiment, the interior of the “tube” can form asingle intracellular chamber, and an “upper” chamber piece can fitaround the tube to provide upper chambers. In this aspect, cells,measuring solution (such as extracellular solution) and compounds can beadded to individual upper chambers that can also contain, or be inelectrical connection with, recording electrodes. The inner tube chambercan be a common chamber that has fluidic and pneumatic connections forproviding measuring solutions and applying pressure for sealing of cellsor particles to ion transport measuring holes. Preferably in thisembodiment the lower chamber comprises or is in electrical connectionwith a reference electrode.

The present invention also includes a method of using a flexible biochipfor measuring ion transport activity or properties. The flexible biochipcan be part of a device in which sections of the flexible biochip aresequentially unwound for sequential sets of assays, or can be used as anat least partly curved surface.

The flexible biochip concept can be applied to not only ion transportassays, but also other high-throughput tests, in which a expanse of thebiochip is used for testing at a time, where the top and optional thebottom surface of the biochip can be engaged in activities such asreagent delivery, detection, separation, etc.

Theta Tubing-Based Chip

Another aspect of the present invention is a method of making amultiplex ion transport measuring device using theta tubing. Eithersemicircular or rectangular theta tubing can be used, however, in somecases rectangular theta tubing can be preferred because the septumbetween the theta openings (referred to herein as “compartments”) istypically of a more uniform thickness in rectangular theta tubing. Inthis method, multiple segments of theta tubing can be stacked on top ofone another or arranged side-by-side, where each segment comprises anion transport measuring means (recording site).

The method comprises: providing at least two segments of theta tubing,each of which comprises an upper compartment and a lower compartment,where the upper compartment and lower compartment is separated by aglass septum; cutting an opening in the top of the theta tubing segmentsto provide access to the upper compartment; using the access at the topof the upper compartment to make at least one hole through the glassseptum that separates the upper and lower compartments of each piece oftheta tubing; and attaching the at least two segments of theta tubingone on top of another, such that the bottom compartment of a secondtheta tubing segment is on top of the upper compartment of a first thetatubing segment.

Preferably, openings cut in the top that are made to provide access forlaser drilling or etching or the hole are sealed prior to stacking thetheta tubing segments on top of one another. FIG. 38A depicts a thetasegment in which a hole has been cut in the top for laser access. Forexample, plastic, rubber, or even glass can be used to close the openingusing adhesives or heat for sealing. For example, the opening in the topof the top compartment can be sealed when the theta segments are stackedon top of one another, preferably by placing a gasket (such as a pieceof flexible rubber, plastic, or silicone) over the opening and stackingthe next theta segment on top of it. The gasket can be held in place byadhesives clamps, or f heat can also be used to attach the stacked unitsto one another. Sealing of the hole can be done such that a port is leftin the top of the top chamber. In embodiments where the units areattached side-by-side, the port can be used for adding compounds orcells.

In the assembled device, each theta tubing segment comprises at leastone (preferably one) ion transport recording site, and each theta tubingsegment comprises an ion transport recording unit, having an upperchamber (upper compartment of the theta tubing segment) and a lowerchamber (lower compartment or opening of the theta tubing segment). Themultiple ion transport measuring units can be arranged vertically (FIG.38B), with the upper and lower chambers of each unit open on eitherside. In an alternative design, multiple ion transport measuring unitscan be arranged side-by-side (FIG. 38C), with the upper and lowerchambers of each unit open each open on either side.

The open sides of each chamber are used to attach conduits for fluidflow, cell and compound delivery, and pneumatic control. In somepreferred embodiments of the present invention, depicted in FIGS. 38Band 38C, individual conduits for providing extracellular solution,compounds, and cells, are attached to one side of each upper compartmentof the theta structure, and individual conduits lead out of each upperchamber at the opposite side of the theta structure. In these designs,individual conduits providing intracellular solution can be attached toone side of each lower compartment of the theta structure, andindividual conduits for outflow of intracellular solution lead out ofeach lower chamber at the opposite side of the theta structure. Pressurecan be applied either from the intracellular inflow conduit or theintracellular outflow conduit.

Many different arrangements are possible for providing solutions,compounds, cells or particles, and pressure to a theta multiplex iontransport measuring device. For example, cells can be introduced to thelower chamber, and pressure for sealing of cells can be applied to theupper chamber. Conduits can be arranged in any way that can providepressure for particle sealing and fluid flow for the addition ofsolutions, compounds, and particles such as cells.

In making the device, commercially available theta tubing can be used.The glass tubing can be cut into segments of any size that will allowthe segment to function as ion transport measuring unit. For example, insome preferred embodiments, the segments can be from about 0.1 mm toabout 80 mm in width, more preferably from about 1 mm to about 10 mm inwidth. The volumes of the upper and lower chambers of the units can bethe same or different. Preferably, the extracellular chamber hasinternal measurements of at least 20 microns by 20 microns, and theintracellular chamber has internal measurements of at least 10 micronsby 10 microns.

Dimensions of the ion transport measuring through holes that are made(for example, by laser drilling or etching) into the theta separatorsegments are preferably from about 0.3 to about 8 microns in diameter.The ion transport measuring holes can also include etched or laserdrilled counterbores, as described previously in this application.

As described herein, the surface of the theta segment can be treated orcoated to promote sealability of the surface as described previously inthis application.

From two to 100 or more theta segments can be attached in vertical orparallel orientation (see FIGS. 38B and 38C). Attachment can be throughthe use of adhesives, gaskets, and the like. As mentioned above, theopening in the upper chamber can be sealed before or during attachmentof the units. Conduits for the addition of solutions, cells, andcompounds, and for the application of pressure can be attached both openends of the chamber in any functional way, and can also use gaskets,adhesive, adaptors, etc. Electrodes, if provided within the chambers,can be inserted into chambers before or after assembling the multiplexstructure.

Electrodes can be situated within upper and lower chambers of thesegments. Alternatively, for a theta multiplex device, an electrodeprovided external to a chamber can be in electronic contact with one ormore upper chambers through an electrolyte (solution) bridge. Forexample, one or more electrodes can be provided in one or more conduitsleading to one or more upper chambers of the device, or provided as partof the ion transport recording machinery (signal source/amplifier) suchthat the electrode or electrodes are in electrical contact with an iontransport measuring solution. Similarly, one or more electrodes can beprovided in one or more conduits leading to one or more lower chambersof the device, or provided as part of the ion transport recordingmachinery (signal source/amplifier) such that the electrode orelectrodes are in electrical contact with an ion transport measuringsolution. In some preferred embodiments of the present invention inwhich a device is used for whole cell ion transport measurement, theupper chamber of each theta ion transport measuring unit is the“extracellular chamber” that comprises or is in electrical contact witha reference electrode. In this case, multiple upper chambers canoptionally be in electrical contact (for example, through conduits thatprovide solution bridges) with a single reference electrode.

Many other electrode arrangements are possible, however, including butnot limited to a single reference electrode in electrical contact withmultiple lower chambers (which can be “intracellular” or “extracellular”chambers of the units), individual reference electrodes for each lowerchamber, individual reference electrodes for each upper chamber, etc.Recording electrodes can also be provided within chambers or inelectrical contact (for example, through conduits that provide solutionbridges) with chambers.

The present invention also includes ion transport measuring devices madeusing the methods of the present invention. These ion transportmeasuring devices comprise at least two attached theta tubing segments,wherein the theta separator segment of each of the theta tubing segmentscomprises an ion transport measuring hole. Preferably, the upper andlower chambers of each theta tubing segment comprises or is inelectrical contact with at least one electrode. Preferably, the thetaion transport measuring device comprises conduits that attach to upperand lower chambers of each theta tubing segment. The theta ion transportmeasuring device can comprise any functional arrangement of electrodesor electrical connections to electrodes, and any functional arrangementof fluidic and pneumatic structures (such as conduits, valves, and canconnect systems for controlling fluid flow and pressure (for example,pumps), and electronic equipment for ion transport measurement.

The present invention also includes methods of using ion transportmeasuring devices comprising at least two attached theta tubing segmentsto measure at least one ion transport activity or property of at leastone particle (such as a cell). Methods of ion transport measurement arewell known in the art and also described herein. The present device canbe used for any type of ion transport measurement, including whole cell,single channel, outside-out patch and inside-out patch recording. Themultiplex theta device can be used for testing the effect of known andunknown compounds on ion transport activity of cells and particles.

Fluidic Systems

The present invention provides novel fluidic systems for deliveringsolutions, compounds, and particles (such as cells) to compartments ofion transport measuring devices. These fluidic systems can in many casesbe applied to a number of chip designs and device designs that may varyin their structures, electrode arrangements, and pressure systems.

Upper Chamber Conduit with Openings for on Transport Recording Sites

One aspect of novel fluidic systems of the present invention is an iontransport measuring device comprising an upper chamber conduit thatcomprises multiple openings each of which accesses a single iontransport measuring hole, schematically depicted in FIG. 39. The devicecomprises: a biochip comprising at least two ion transport measuringholes; at least one conduit on the upper side of said biochip, in whichthe conduit comprises at least one opening for each of said at least twoion transport measuring holes; at least one inlet; and at least oneoutlet. Each conduit can have more than one opening. Preferably, thechip of the device comprises 2 or more holes, more preferably 16 ormore, more preferably yet 48 or more, and most preferably 96 or more. Ina variation, the conduits comprise interfacing piezo actuators that canchange the solutions dispensed over the ion transport measuring sitesvery quickly.

Different compounds and/or solutions can be delivered to the recordingsite through these channels. Measuring solutions (such as ES) and cellsor compounds can be added at the channel inlet. A reference electrode isprovided within or in electrical connection with the upper chamber. Inone embodiment of this aspect of the present invention, there areadditional openings at the top of the upper chamber channel over the iontransport recording sites. Particles such as cells can be added throughthese openings. Interfacing piezo actuators that can change thesolutions dispensed over the ion transport measuring sites very quicklycan also be localized to opening over the ion transport recording sites.Preferably, a lower chamber piece engages the ion transport measuringdevice, where the lower chamber piece preferably comprises individualchambers, such that each ion transport measuring hole of the biochipaccesses its own lower chamber. The lower chambers preferably compriseor are in electrical connection with individual recording electrodes andhave connections to a pump or other pressure-generating device, andconduits for the addition and removal of measuring solution.

In using this type of device, a single cell type can be added to thistype of device for screening different compound solutions.Alternatively, different cell types or particles comprising differention transports can be added at different ion transport recording sites.Immediately after cell addition, pressure applied from the bottom of thechip can allow the cells (or other particles) to seal at ion transportmeasuring holes. In this way, a particular ion transport recording sitehas particular type of cell or particle sealed to it. Compounds can beadded through the channel inlet, and ion transport recordings cansimultaneously measure the response of various cell types or ion channeltypes to a given compound. Optionally, the upper chamber channel can beflushed to remove the compound of interest, and a second compound can beadded by pushing or pumping a second compound-containing solution intothe channel. In this way, multiple compounds (or differentconcentrations of one or more compounds) can be assayed for theireffects on one or more cell types or one or more ion transport types.

Alternatively, the solution—delivering channels can be tips such aspipette tips, in this case, solutions or compounds are filled into thetips by suction, and delivered out to the recording sites by positivepressure through the same tip.

In other related designs, the upper chamber channel may not haveopenings localized over ion transport recording sites, and after theaddition of particles such as cells, a compound-containing solution canbe added through the channel inlet. After ion transport measurement, thechannel is flushed with solution lacking compound, and then a newcompound-containing solution can be added through the channel inlet. Inthis case, replica assays can be conducted simultaneously using the sametype of cell or ion transport.

The present invention includes ion transport measuring devices thatinclude upper chamber channels with openings at multiple ion transportrecording sites, including devices that comprise ion transport chips asthey are known in the art and described herein, including, for example,planar chips, flexible chips, and MCP chips. Where feasible, chips usedin the devices having upper chamber channels can be treated, such asusing methods described herein, to improve their sealing properties.

The present invention also includes methods of using ion transportmeasuring devices that include upper chamber channels with openings atmultiple ion transport recording sites to measure ion transport functionand properties. In preferred embodiments, the methods are highthroughput.

One use of such conduits is to deliver tiny amount of compounds in dropsto cells already sealed to the recording sites in low volume ofsolutions. In this case, the surface of the chip can comprise ahydrophobic material, with the exception of the surface immediatelysurrounding ion transport measuring holes or microwells, to reduce fluidflow between ion transport recording sites in the absence of directedfluid flow through the chamber, for example, by coating chips with ahydrophobic material as described above in Aspect 21. Fusion of thecompound drop and the small volume of solutions at the recording sitesallows for fast and efficient compound delivery. The fused drops willnot fuse together to cross-contaminate recording sites since the dropsare bounded by the hydrophobic coating. Wash out can be achieved byflushing the entire upper chamber with wash solution and subsequentremoval of wash solution. After washout, recording sites are ready toreceive the next delivery of compounds.

Upper Chamber Channel Strips

Yet another aspect of the present invention is an ion transportmeasuring device comprising a biochip that comprises multiple iontransport measuring holes, in which the device has at least one upperchamber channel that has access to two or more of the multiple iontransport measuring holes. The device also has at least one lowerchamber channel that that has access to two or more of the multiple iontransport measuring holes. The one or more upper chamber channel stripsare arranged approximately perpendicularly to the at least one lowerchamber channel. The device also comprises a compound delivery systemfor delivering compounds or solutions to individual ion transportmeasuring sites.

In preferred embodiments, the device has multiple upper chamberchannels, each of which has access to two or more ion transportmeasuring holes, and multiple lower chamber channels, each of which hasaccess to two or more ion transport measuring holes. The upper channelsextend in the m direction, and the lower channels extend in the ndirection, such that each ion transport measuring hole that contacts aparticular upper channel strip (for example, M₁) contacts a differentlower channel (for example, lower channel N₁, N₂, N₃, N₄, N₅, or N₆),and each ion transport measuring hole that contacts a particular lowerchannel (for example, N₁) contacts a different upper channel strip (forexample, upper channel strip M₁, M₂, M₃, M₄, M₅, or M₆).

In some preferred embodiments, each upper channel strip comprises, or isin electrical contact with, an independent recording electrode, and eachlower channel comprises, or is in electrical contact with, anindependent reference electrode. Alternatively, each upper channel stripcan comprise, or be in electrical contact with, an independent referenceelectrode, and each lower channel can comprise, or be in electricalcontact with, an independent recording electrode.

In preferred embodiments, the upper channel strips are used asextracellular chambers that, during ion transport assays, comprisemeasuring solution, cells (or other particles), and, preferably,compounds to be tested. Various designs can be used that allowindividual compounds (or compound concentrations) to be localized toindividual recording sites within an upper chamber channel. For example,the “piped in” compound delivery system described above, in whichindividual conduits (pipes) deliver compounds directly over an iontransport measuring site can be applied to this channel matrix device.Preferably, however, peizo controlled tips deliver compounds directlyover an ion transport measuring site. In this case, the uppermicrofluidics channels fill by hydrophilicity to wet all individualrecording sites with measuring solution. Preferably, the biochip hasdepressions or microwells at the ion transport recording sites thatpromote retention of solution (such as recording solution andcompound-containing solution) at the ion transport recording sites. Theupper chamber channels have a low diameter to length ratio that retardsmixing, and the compounds are delivered to individual recording sitessimultaneously so as to prevent hydrostatic pressure from forcing themto flow into adjacent chambers.

Before or after compound delivery, ion transport recording takes placeby sequentially recording using reference electrode 1 (in combinationwith all possible recording electrodes), then using reference electrode2 (in combination with all possible recording electrodes), then usingreference electrode 3 (in combination with all possible recordingelectrodes), etc., and optionally return to electrode 1 for a nextcycle.

In a related aspect, upper chamber channel strips can contact iontransport recording sites along the upper side of an ion transportmeasuring chip, and each ion transport recording site can have anindependent lower chamber that comprises or is in electrical contactwith a recording electrode. Preferably, the biochip has depressions ormicrowells at the ion transport recording sites that promote retentionof solution (such as recording solution and compound-containingsolution) at the ion transport recording sites. A common referenceelectrode is present in the upper chamber channels, in which theelectrode is a narrow strip of conductive material (for example, metal)built along the surface of the interconnected upper chamber channels.The electrode strip is coated along its upper surface with anonconductive material (for example, a polymer), and is exposed tosolution only at recording sites. In this way the upper chamber channelsremain in electrical communication, but fluid communication between iontransport measuring sites is minimized.

The present invention includes ion transport measuring chips thatcomprise upper chamber channels, and devices comprising ion transportmeasuring chips that comprise upper chamber channels. The chamber stripsconcept can be applied together with the various chip designs, includingfor example, the flexible biochips described above that provide the iontransport measuring holes.

The present invention also includes methods of using includes iontransport measuring chips that comprise upper chamber channels tomeasure ion transport activity and properties of particles such ascells.

Compound Delivery into Upper Chamber by Fluidic Pipes

The invention also includes devices that comprise a substrate thatcomprises at least two ion transport measuring holes (ion transportmeasuring chip); at least two lower chambers that engage the iontransport measuring chip, such that each or the at least two iontransport measuring holes is in register with an individual lowerchamber; at least one upper chamber that has fluid access to the atleast two ion transport measuring holes; and at least two conduits or“pipes” that can be positioned over the at least one upper chamber,where each of the at least two conduits aligns directly over and inclose proximity to an ion transport measuring hole (see, for example,FIG. 40).

The device can have a single common upper chamber, or can have a seriesof upper chamber channels that may or may not connect with one another.Where they are non-interconnecting channels, more than one referenceelectrode and more than one inflow and outflow conduit is required.

In preferred embodiments, an upper chamber comprises a referenceelectrode, and is connected to inflow and outflow conduits such thatfluid flow of measuring solution can be established through the upperchamber. Cells can be added through the inflow conduit, and aftersealing of cells at ion transport measuring sites, compounds can beadded at individual recording sites through the fluidic pipes. Inpreferred embodiments, the ion transport measuring chip comprises anarray of ion transport measuring holes, and an array of fluidic pipescan be moved over the chip. The structure supporting the pipes engages astructure on the chip that precisely aligns and gauges the travel of thetubes so that they are positioned just over the recording apertures andcan provide concentration clamping of delivered compounds in theimmediate vicinity of the cell.

The pipes can deliver tiny amount of compounds in drops to cells alreadysealed to the recording sites in low volume of solutions, such as thosein the microwells of hydrophobic chip. Fusion of the compound drop andthe small volume of solutions at the recording sites allow for fast andefficient compound delivery. The fused drops will not fuse together tocross-contaminate since the drops are bounded by hydrophobic coatings.Wash out can be achieved by flushing the entire upper chamber with washsolution and subsequent removal of wash solution. The recording sitesare now ready to receive the next delivery of compounds.

The present invention also includes methods of using ion transportmeasuring devices that comprise pipe arrays for delivering compounds ation transport measuring sites of upper chambers. In broad outline, suchmethods include: providing measuring solution in the lower chambers ofthe device; providing particles in measuring solution in an upperchamber of the device; sealing particles at ion transport measuringholes; providing continuous flow of measuring solution through the upperchamber; halting the flow of measuring solution through the upperchamber; positioning an array of pipes over the upper chamber;delivering compounds continuously at recording sites through the pipes,and measuring ion transport function or properties. The upper chamber ofthe ion transport measuring device can optionally be flushed after iontransport measurement, and optionally new compounds can be added toupper chamber recording sites using the pipe array. The process can berepeated multiple times.

Nozzle Delivery of Compounds

The invention also includes devices that comprise a substrate comprisingat least two ion transport measuring holes; an upper chamber thatcomprises or is in electrical contact with at least one referenceelectrode, in which the upper chamber accesses the two or more iontransport measuring holes; and a compound delivery system that candeliver compound or solution to each ion transport measurement siteindividually, in which the compound delivery system comprises a fluidicsblock that comprises funnel structures terminating in outflow nozzlesthat can be aligned over the chip such that a single nozzle ispositioned over each ion transport recording site (see FIG. 41).Preferably, the device comprises or engages at least two lower chambersin register with the two or more holes of the chip. During ion transportmeasurement, each of the individual lower chambers preferably comprisesor is in electrical contact with a recording electrode.

When the fluidics block is aligned over the chip, the outflow nozzlesare positioned close to the surface of the measuring solution of theupper chamber, but not in contact with it. Preferably, the nozzle is atthe end of a funnel structure, the nozzle diameter is greater than tentimes the diameter of the cells, and the funnel size is large enough toallow the compound solution within it to flow out of the nozzle oversufficient time that more compound solution can be delivered to thefunnels (such as by dispensing pipette tips) without creating bubbleswithin the funnel or nozzle area.

In using the device, lower chambers are filled with measuring solutions,and the the upper chamber is filled with measuring solution and cells(or other particles) are added. Cells (or other particles) are sealed tothe ion transport measuring holes by applying suction to the lowerchambers. By controlling fluid flow through the upper chamber, an evenbut shallow bath is produced that has continuous flow. Optionally theinflow into the upper chamber will be by microfluidics channels openingnear each aperture. The fluidics block for compound addition ispositioned over the holes on the chip such that the outflow nozzles areclose to the surface of the measuring solution in the hole within theupper chamber, but not in contact with it. After control currents arerecorded, compounds are added to the fluidics block from above. As longas the block is held above the measuring solution of the upper chamber,the compounds do not flow beyond into the upper chamber due to thesurface tension of the small droplets that form at the bottoms of thenozzles. The voltage protocol sweeps are coordinated such that once thedrugs are requested, the entire block may be lowered to contact theupper chamber measuring solution, and brought down to within half of anozzle width of the cells such that the drug-containing solution isallowed to flow out of the nozzles by gravity to clamp the concentrationof drug in the immediate vicinity of the cells.

The present invention includes ion transport measuring devices thatinclude a biochip comprising ion transport measuring holes and acompound delivery system that can deliver compound or solution to eachion transport measurement site individually using a fluidics block thatcomprises outflow nozzles that can be aligned over one or more upperchambers of a chip such that a single nozzle is positioned over each iontransport recording site. In preferred embodiments, the devices arehigh-throughput devices that comprise at least 48, at least 96, or atleast 384 ion transport measuring sites and a multiplicity of nozzlesfor dispensing compounds at the ion transport measuring sites.Preferably, the number of dispensing nozzles of the fluidics block isequal to the number of ion transport measuring sites of the iontransport measuring chip.

The devices of the present invention can comprise ion transport chips asthey are known in the art and described herein, including, for example,planar chips, flexible chips, and MCP chips. Where feasible, chips usedin the devices having upper chamber channels can be treated, such asusing methods described herein, to improve their sealing properties.

Chamber Separator

In yet another aspect of the present invention, an ion transportmeasuring device comprises a substrate comprising at least two iontransport measuring holes; a common upper chamber, and a physicalseparator unit that can be lowered onto the chip to separate the commonupper chamber into multiple individual chambers. Preferably, the devicecomprises or engages one or more lower chambers.

The physical separator unit can be lowered into the upper chamber (whichcan be in the form of a tank with ion transport measuring holes in thebottom) after measuring solution and cells (or other particles) havebeen introduced into upper chamber and preferably after particles havebeen sealed to the ion transport measuring holes. The physical separatorcan reversibly fasten on to the substrate. The physical separator cancomprise any fluid-impermeable material, and preferably comprises acompressible material where it contacts the surface of the chip to formseals against the chip so that the attached separator formsfluid-impermeable separated channels. For example, the separator unitcan comprise multiple electrodes, each of which can contact a separatechamber when the separator engages the chip. In this case, sealing canoccur after the separator has been positioned to form the chambers.

The device can also engage a lower chamber that can comprise a referenceelectrode.

After cells have sealed to the chip and the separator unit has formedseparate upper chambers, compounds can be added to the individual upperchamber, either by conduits or fluid dispensing systems. Ion transportrecording can then be performed using upper chamber recording electrodesand a bottom chamber reference electrode.

It is also possible to have multiple lower chambers that engage thechip. In this case the lower chambers can optionally have recordingelectrodes. In this case the upper chambers can comprise or be inelectrical contact with independent reference electrodes, oralternatively, a common electrode (for example, an electrode thattraverses the upper surface of the chip) can contact or be brought intoelectrical contact with the multiple upper chambers and be used as areference electrode.

The devices of the present invention that comprise physical separatorunits for forming chambers can comprise ion transport chips as they areknown in the art and described herein, including, for example, planarchips, flexible chips, and MCP chips. Where feasible, chips used in thedevices having upper chamber channels can be treated, such as usingmethods described herein to improve their sealing properties.

Pneumatic and Electronic Matrix

In a related aspect, the present invention includes an ion transportmeasuring device comprising: a biochip that comprises multiple iontransport measuring holes, in which the device has at least one upperchamber channel that has access to two or more of the multiple iontransport measuring holes. The device also has at least one lowerchamber channel that that has access to two or more of the multiple iontransport measuring holes, where each lower chamber channel has separatepneumatic connections for generation pressure for sealing particles ation transport measuring holes.

The one or more upper chamber channels are arranged approximatelyperpendicularly to the at least one lower chamber channel. The devicealso comprises a compound delivery system for delivering compounds orsolutions to individual ion transport measuring sites.

In preferred embodiments, the device has multiple upper chamberchannels, each of which has access to two or more ion transportmeasuring holes, and multiple lower chamber channels, each of which hasaccess to two or more ion transport measuring holes. The upper channelsextend in the m direction, and the lower channels extend in the ndirection, such that each ion transport measuring hole that contacts aparticular upper channel (for example, M₁) contacts a different lowerchannel (for example, lower channel N₁, N₂, N₃, N₄, N₅, or N₆), and eachion transport measuring hole that contacts a particular lower channel(for example, N₁) contacts a different upper channel (for example, upperchannel strip M₁, M₂, M₃, M₄, M₅, or M₆).

In preferred embodiments, the upper channels are used as extracellularchambers that, during ion transport assays, comprise measuring solution,cells (or other particles), and, preferably, compounds to be tested.Various designs can be used that allow individual compounds (or compoundconcentrations) to be localized to individual recording sites within anupper chamber channel. For example, the “piped in” compound deliverysystem described in Aspect 26, above, in which individual conduits(pipes) deliver compounds directly over an ion transport measuring sitecan be applied to this channel matrix device. Preferably, however, peizocontrolled tips deliver compounds directly over an ion transportmeasuring site. Preferably, the biochip has depressions or microwells atthe ion transport recording sites that promote retention of solution(such as recording solution and compound-containing solution) at the iontransport recording sites.

Immediately following compound delivery, ion transport recording takesplace by sequentially recording using by applying pneumatic pressure tolower channel 1 to seal particles at ion transport holes that accesslower channel 1, then by applying pneumatic pressure to lower channel 2to seal particles at ion transport holes that access lower channel 2,then by applying pneumatic pressure to lower channel 3 to seal particlesat ion transport holes that access lower channel 3, etc.

Device having Compound Delivery Plate

Yet another aspect of the present invention is an ion transportmeasuring device that comprises a substrate comprising at least two iontransport measuring holes, at least two upper chambers in register withthe two or more ion transport measuring holes; at least two lowermicrowells, each of which is positioned around an ion transportmeasuring hole, and each of which is connected to a common lower chamberchannel; and a compound delivery plate, in which the compound deliveryplate has drug delivery sites in register with the lower microwells,where the compound delivery plate can reversibly come into contact withthe lower microwells. In this design, depicted in FIG. 42, the two ormore upper chambers are connected to a pneumatic system for sealingcells to the ion transport measuring holes on the lower side of thesubstrate and each of the upper chambers comprises or is in electricalcontact with an individual (recording) electrode.

Electrical and pneumatic connection to the upper wells of the iontransport measuring chip can be provided by a separate adaptor plate.Preferably, each independent upper well connects to a separate recordingelectrode.

The lower channel (which accesses the lower microwells) is connected toconduits to provide fluid flow of measuring solution through thechannel. The bottom surface of the chip, with the exception of themicrowell surfaces, is preferably hydrophobic to aid in maintainingfluid separation between microwells when fluid is removed from the lowerchamber. A reference electrode can preferably be provided on the lowersurface of the chip, connected to the compound delivery plate, or inelectrical contact with the lower channel.

In some preferred designs, at the time of operation of the device, thedrug delivery sites have compounds spotted, or printed on them in dropsor dried form. In some other preferred designs, the drug delivery sitesof the compound delivery plate are apertures through which droplets ofcompound-containing solution can be pushed up from below.

In operation, measuring solution is added to the upper chambers, andmeasuring solution and cells are introduced into the lower chamberchannel through conduits. Pressure is applied to the upper wells (eitherindividually or commonly connected to pressure controls) to pull cellsup from the lower channel into the lower microwells and seal themagainst the ion transport measuring holes of the chip. Sealing occurs inthe presence of complete solution superfusion of the bottom chamber.After the seals have formed, solution is removed from the channel, withthe exception of the microwells, which in the case of coated surfaceelectrodes, are in electrical connection with the electrodes. At thistime compounds are applied to the microwells as the delivery plate isbrought into contact with the lower surfaces of the microwells. Iontransport measurement can then be performed.

The same device can be used in inverted orientation, with cells sealingto the top of the chip, and the compound delivery plate is positionedabove the chip to apply compounds from the top side of the chip.

Devices Featuring Multichannel Pipeting Delivery of Compounds

Yet another aspect of the present invention is an ion transportmeasuring device that comprises a substrate comprising at least two iontransport measuring holes; at least two upper microwells each of whichis positioned around an ion transport measuring hole, and each of whichis connected to a common upper chamber channel; at least one lowerchamber; and a compound dispensing system that can dispense compoundsolution into the upper microwells. In this design, the one or morelower chambers are connected to a pneumatic system for sealing cells tothe ion transport measuring holes on the upper side of the substrate.

The upper channel (which accesses the upper microwells) is connected toconduits to provide fluid flow of measuring solution through thechannel. The top surface of the chip, with the exception of themicrowell surfaces, is preferably hydrophobic to aid in maintainingfluid separation between microwells when fluid is removed from the upperchamber.

The compound delivery system can comprise conduits or fluid dispensingtips, for example, of a multichannel pipeting system, that can bearranged in an array that can align with the array of microwells. Thecompound delivery system can deliver compound solution over themicrowells in droplets that localize to the microwells and do not spreadto other wells due in part to the hydrophobicity of the chip uppersurface, as depicted in FIG. 43. Preferably, the compound drops are verylarge compared to the microwell volume, so that there is little compounddilution when it is delivered.

Various electrode arrangements can be used with this device. Forexample, the upper surface of the chip can comprise a common referenceelectrode that is coated with a hydrophobic material except where itcontacts the individual microwells. In this case, the device hasmultiple independent lower wells, each of which is associated with asingle ion transport measuring hole. Each lower well comprises or is inelectrical contact with an independent electrode that can be used forion transport recording. The lower well electrodes can optionally beprovided by a separate, replaceable adaptor plate that can alsooptionally comprises connections to pneumatic devices for pressurecontrol. In an alternative, the device can have a single bottom chamberthat comprises a reference electrode. Individual recording electrodescan be provided in connection with the upper microwells. For example,the recording electrodes can be attached to the compound deliverysystem, such that positioning of the compound delivery system over themicrowells can also serve to position and dip an electrode into themicrowell.

In operation, measuring solution is added to the lower chambers, andmeasuring solution and cells are introduced into the upper chamberchannel through conduits. Pressure is applied from the bottom of thechip to seal cells against the ion transport measuring holes of thechip. Sealing occurs in the presence of complete solution superfusion ofthe upper chamber. After the seals have formed, solution is removed fromthe upper channel, with the exception of the microwells, which in thecase of a coated surface electrode, are in electrical connection withthe reference electrode. At this time compounds are applied to themicrowells by position the compound delivery system over the biochip anddispensing compound drops over the microwells. Ion transport measurementcan then be performed on the cells sealed at the microwells.

The lower well electrodes can optionally be provided by a separate,replaceable adaptor plate that can also optionally comprises connectionsto pneumatic devices for pressure control. This adaptor plate design canbe applied to all designed for biochips, fluid delivery mechanisms, aswell as electrical and/or pneumatic control systems known in the art ordescribed in this entire application.

Further Embodiments

The present invention includes chips, devices, and methods for iontransport measurement that can be used to efficiently assay testparticles such as cells. The devices allow ion transport assays that canbe used in a variety of ion transport measurement applications,including but not limited to determining the effects of compounds (suchas compounds of interest or test compounds) on ion transport activity.The assays can also be used to assess cell condition, “sealability”,responsiveness to compounds or treatments before performing a battery oftests using the cells, or to rapidly determine the effects of growthconditions, developmental stages, hormone responsiveness on the iontransport activity of cells. In some embodiments, the ion transportmeasuring devices can be used for other assays in addition to iontransport measurement assays. In some embodiments, the ion transportmeasuring devices can designed such that cells in a chamber of thedevice can be microscopically viewed before, during, and/or immediatelyafter an ion transport measurement assay.

Another aspect of the present invention is a device for ion transportmeasurement that comprises a chip having at least one ion transportmeasuring hole and an upper chamber piece comprising at least onechamber, where the one or more upper chambers comprise at least twoopenings, and where one of the two or more openings is on one side ofthe one or more ion transport measuring holes and the other of the twoor more openings is on the other side of the one or more ion transportmeasuring holes. In this device, the upper chamber is a “flow-through”perfusion chamber that accesses at least one ion transport measuringhole (depicted in FIG. 44).

In some embodiments of this device, one of the two or more openingscomprises a reservoir at its end where cells and, potentially, compoundscan be added to the upper chamber such as by a fluidic system orpipette.

The upper chamber can comprise an electrode, or, during use of thedevice, can be in electrical contact with an electrode that can be partof the signal amplifier machinery, or can be provided in tubing leadingto the chamber.

In preferred embodiments, the device has a single upper chamber with twoupper chamber openings, one on either side of the one or more iontransport measuring holes, such that measuring solution buffers, orcompound containing solutions (such as Extracellular Solution, ES) canflow through the upper chamber. For example, measuring solution can bepumped through the upper chamber to fill or wash the chamber. Inpreferred embodiments in which one opening comprises a reservoir at itsend, particles such as cells and compounds can optionally be added viathe reservoir. In the alternative, either particles, compounds, or bothcan be added to the upper chamber at an opening that is not connected toa reservoir. Preferably, the upper chamber of the flow-through upperchamber device is transparent, so that cells in the upper chambers canbe viewed microscopically.

In using the device, the upper chamber of the flow-through upper chamberdevice can engage a lower chamber piece. The lower chamber piece can bein the form of a tray or tank, and preferably has at least one inlet andat least one outlet for allowing measuring solution (such as IS,intracellular solution) to flow into the chamber and for the applicationof pressure for sealing particles to the one or more ion transportmeasuring holes. In some preferred embodiments, such as that depicted inFIG. 45, the lower chamber is also a single flow-through channel, withan opening at one end for the introduction of solutions such asmeasuring solution, and an opening at the other end for outflow ofsolutions, and preferably, connection to pneumatic devices for applyingpressure to seal particles to the one or more ion transport measuringholes.

The present invention also includes ion transport measuring devices withflow-through upper chambers that are reversibly or irreversibly attachedto lower chamber pieces, such as, for example, the single flow-throughchannel lower chamber piece described above and depicted in FIG. 45. Inthe preferred embodiment of FIG. 45, the device comprises a biochiphaving a single ion transport measuring hole, a flow-through upperchamber and a flow-through bottom chamber, where the cells can be viewedthrough the top of the upper chamber using a microscope.

Another aspect of the present invention is an ion transport measuringdevice comprising a chip having at least two ion transport measuringholes, an upper chamber piece comprising a single upper chamber; and alower chamber piece comprising two or more isolated bottom chambers(depicted in FIG. 46). Preferably, each lower chamber is connected in atleast two conduits for inflow and outflow of measuring solution (such asIS) and the application of pressure for sealing cells. Preferably, theupper chamber is connected in at least two conduits for inflow andoutflow of measuring solution and for the addition of cells and,optionally, compounds. In the alternative, the upper chamber can haveopenings (for example, at the top) where solutions, cells, andoptionally compounds can be added. The common upper chamber can be open,or can have a transparent top so that cells in the chamber can be viewedmicroscopically or fluorescence measurement can be performed from thetop.

Preferably, during operation of the device each of the lower chamberscomprises or is in electrical contact with an individual electrode.Preferably, during operation of the device the upper chamber comprisesor is in electrical contact with a common electrode.

Simple Chip

A further aspect of the present invention is a glass chip comprising atleast one ion transport measuring hole (see, for example, FIG. 47). Thechip can be single use, and can insert into or assemble with structuresthat comprise upper chamber pieces and lower chamber pieces.

The chip can comprises any solid material such as metals, ceramics,polymers, inorganic and organic hybrid materials, plastics, silicondioxide, or glass, and the ion transport measuring holes can be etched,laser drilled, cut, punched out, or bored into the material. Inpreferred embodiments, the chip is a glass chip and the ion transportmeasuring holes are laser drilled. Preferably, the chip issurface-treated, such as by using methods described herein.

Preferably, during use, the chip is inserted into or assembled with anupper chamber piece having isolated upper chamber wells and a lowerchamber piece having isolated lower chamber wells, but other designs arepossible. For example, the chip can be inserted into or assembled withan upper chamber piece having isolated upper chamber wells and a lowerchamber piece having a common lower chamber well, or can be assembledwith an upper chamber piece having a common upper chamber well and alower chamber piece having isolated lower chamber wells.

In preferred embodiments, the chip is single-use and disposable, and theupper and lower chamber pieces, as well as associated electrodes (whichcan be part of the signal amplifier machinery or electrodes that can beattached or connected to the wells), are reusable.

Devices Comprising Chips having Wells made of Compressible Material

In a related aspect, the present invention comprises a chip comprisingat least one ion transport measuring hole, in which the chip comprisesat least one upper well on its top surface that comprises a layer ofwax.

The chip can comprises any hard material such as metals, ceramics,polymers, inorganic and organic hybrid materials, plastics, silicondioxide, or glass, and the ion transport measuring holes can be etched,laser drilled, cut, punched out, or bored into the material. Inpreferred embodiments, the chip is a glass chip and the ion transportmeasuring holes are laser drilled. Preferably, the chip issurface-treated, such as by using methods described herein.

Preferably, the wax on the upper surface of the chip forms individualwells, after a single ion transport measuring hole is created on the topsurface of the chip (see, for example, FIG. 48). As in the previousembodiment, during use, the chip is assembled with one or morestructures to form an ion transport measuring device. In this case,however, the chip comprises upper chamber wells on its surface, and thechip engages a structure that preferably comprises as upper piece topsurface that forms the top of the upper wells, as well as a lowerchamber piece. The wax-formed upper chamber structures on the uppersurface of the chip are at least somewhat compressible, allowing sealingof the upper chamber structures to the upper piece surface when thedevice is assembled.

The upper piece top surface that engages the chip can also includeconduits and, optionally, electrodes that can connect with the one ormore upper chambers of the device when the device is assembled.

Preferably, the chip comprises multiple wax-formed upper chambers andthe lower chamber piece it assembles with has multiple isolated lowerchamber wells, but other designs are possible. For example, the chip canhave a single wax-formed upper chamber, and can be assembled with astructure that comprises multiple isolated lower chambers. In analternative, the chip comprises multiple wax-formed upper chambers andthe lower chamber piece has a single common lower chamber.

In preferred embodiments, the chip having wax-formed upper chambers issingle- use and disposable, and the lower chamber piece and thestructure that comprises the upper piece top surface, as well asassociated electrodes (which can be part of the signal amplifiermachinery or electrodes that can be attached or connected to the wells),are reusable.

Another material that can be used for forming wells on the surface of abiochip is any photoresist, such as SU-8. SU-8 is a photo-curable epoxyoligomer, commonly used for computer chip manufacture. To make one ormore wells on the surface of a chip using SU-8, the liquid form of theoligomer is distributed on the surface of the chip. A mask is used topattern one or more wells. Light induces polymerization of SU-8 in areasnot covered by the mask. After polymerization, the unpolymerized SU-8 iswashed away to leave chamber walls that comprise SU-8 polymer.

Chip with O-Ring Upper Chambers

In a related aspect of the present invention, a chip comprising at leastone ion transport measuring hole is provided with at least one O-ringthat forms an upper chamber around the at least one ion transportmeasuring hole (see, for example, FIG. 49). The chip having O-ring upperchambers can be assembled with at least one structure to form an iontransport measuring device.

The chip can comprises any hard material such as metals, ceramics,polymers, plastics, silicon dioxide, or glass, and the ion transportmeasuring holes can be etched, laser drilled, cut, punched out, or boredinto the material. In preferred embodiments, the chip is a glass chipand the ion transport measuring holes are laser drilled. Preferably, thechip is surface-treated to increase its sealing properties, such as byusing methods described herein.

To assemble an ion transport measuring device, the chip preferablyengages a structure that preferably comprises as upper piece top surfacethat forms the top of the upper wells that can be reversibly attached tothe top of the chip, as well as a lower chamber piece that can bereversibly attached to the bottom of the chip. The upper chamber O-ringstructures on the upper surface of the chip are at least somewhatcompressible, allowing sealing of the upper chamber structures to theupper piece surface when the device is assembled. The O-ring can also besealed to the top of chip surface using an adhesive.

The upper piece surface can also include conduits and, optionally,electrodes that can connect with the one or more upper chambers of thedevice when the device is assembled.

Preferably, the chip comprises multiple O-ring upper chambers and thelower chamber piece has multiple isolated lower chamber wells, but otherdesigns are possible. For example, the chip can have a single O-ringupper chamber, and can be assembled with a structure that comprisesmultiple isolated lower chambers. In an alternative, the chip comprisesmultiple O-ring upper chambers and the lower chamber piece has a singlecommon lower chamber well.

In preferred embodiments, the chip having O-ring upper chambers issingle-use and disposable, and the upper piece surface and lower chamberpiece, as well as associated electrodes (which can be part of the signalamplifier machinery or electrodes that can be attached or connected tothe wells), are reusable.

Method for Performing Excised Patch Voltage Clamp Recordings

Excised voltage clamp recordings such as inside-out or outside inconfigurations as known in the art of voltage clamp studies can beperformed by any planar or non-planar electrode configurations known inthe art, or described in this application or previous applications. Thisis done by adding magnetic beads labeled with antibody(s) against commoncell surface markers after the cell is sealed to the ITM sites;incubation to allow for bead binding to the cell surface; and applyingmagnets to the beaded sealed cells from the open access. Magnetic forceswill remove the beads, together with associated cell membrane, whichallows the formation of “excised patch” configuration at the iontransport measuring sites for single channel or macropatch recordings.

Method of Shipping Ion Transport Measuring Chips

The present also provides methods for shipping ion transport measuringchip and devices in which the upper and lower chambers of the devices orchips are pre-filled with an ion transport measuring solution. Forexample, where the devices are intended for use in performing iontransport measurement assays on whole cells, the devices can be packedwith upper and lower chambers filled with intracellular solution (IS).This can reduce the time required to perform an assay, and also canreduce the complexity of the machinery that interfaces with the deviceand provides fluidic controls and conduits, since the machinery does notneed to add measuring solution to, for example the lower chambers of adevice, but instead can simply flush the upper chamber with anappropriate measuring solution such as extracellular solution (ES) priorto adding cells. This increases the efficiency and reduces the timeneeded for assays, such as high throughput screens. (In cases such asthat described in Aspect 28, where cells are distributed in lowerchambers, the machine flushes the lower chamber with, for example, ES,prior to adding cells.)

The devices or chips can be shipped in blister packs that lock in themeasuring solution, and the entire assembly can optionally be keptrefrigerated until use. The measuring solution used can be specializedfor different types of ion transport assays, different cell types, andthe like. The measuring solution can also be simplified for more generaluse with more than one cell or assay type.

To use devices shipped in measuring solution, after flushingextracellular solution through and adding cells to the one or more upperchambers, a vacuum can be applied to the one or more intracellularchambers that already contain IS to seal cells to ion transportmeasuring holes.

Ion Channel Chip Having Chemical Surface Modification on Plastics andMethods of use

Another aspect of the present invention is an ion channel chip havingchemical surface modification on plastics, such as a plastic surface orsubstrate, and methods of use. Preferably, the plastic surface ismodified to improve the chip's ability for form tight seals for iontransport measurement. Preferably, plastic surfaces can be plasmaetched, which makes the surface clean and creates chemical functionalgroups that can be used for further chemical reactions and/orpolymerizations to provide functionalities on the surface. Thesemodifications are preferably made after holes are provided on the chip,such as through laser drilling. A variety of formats of holes can beprovided, preferably in a standard format or footprint, such as between1 and 1536 holes or more on a chip. These chips can be used in methodsof determining ion channel activities, including high throughputmethods.

In one embodiment the ion channel chip includes a substantially planarpolymer plastic substrate treated with an ionized gas, at least onehole, also referred to as an aperture, positioned substantiallyperpendicular to the plastic substrate and a gasket positioned about theaperture defining the perimeter of a chamber. The ionized gas treatmentmay include exposure to a gas such as plasma gas. Preferably the ionizedgas treatment forms at least one functional group or moiety positionedsufficiently close to the aperture such that a cell can simulataneouslycontact the functional group or moiety and the aperture. This permitsand enhance seal between the chip and a cell.

Chips of the present invention are preferably planar or substantiallyplanar in configuration and made of thin plastic, also referred to aspolymer plastic, material. The plastic material can be any that can belaser drilled to form a hole, also referred to as an aperture orthroughbore, useful for ion transport measurement.

The plastic material chosen for the chip can be any appropriate plasticmaterial, including but not limited to polyimide (such as but notlimited to Kapton™), polyolefin or copolymers thereof (such as but notlimited to Zeonor™), polyacrylate, silicon containing polymer, wax,paraffin wax, Parafilm™, fatty acid based polymers, acrylics or the likeand combinations thereof or copolymers thereof. The plastic materialsare generally characterized as being thin films between 5 and 300 inthickness, preferably a thin film that is rigid or flexible.

The laser and its wavelength can be chosen based on the characteristicsof the plastic, such as the material, thickness, transparency,opaqueness, color and energy or electromagnetic absorptive or reflectivecharacteristics. A variety of lasers are commercially available, such asExcimer Excitation, MO:YAG excitation and Argon and/or Kryptonexcitation lasers. Preferably lasers are in the visible, UV or infraredspectrum. The choice of laser, and the power of the laser to be used,that matches a candidate chip material can readily be determined by testdrilling a material with one or more lasers and determining theeffectiveness and desirability of the resulting hole. For opaquematerials, such as but not limited to plastics, lasers preferably in thevisible spectrum are used. Solid state lasers or copper vapor lasersdescribed herein or known in the art preferable. Preferred laserwavelengths are between about 157 nanometer and about 590 nanometers,although others can be use. Such determination can be made using visualinspection, such as through microscopy, including optical and electronmicroscopy. The resulting hole and surrounding surface is preferably thesize, shape and texture that are appropriate for use in ion transportmeasurement methods. The resulting surface surrounding or generallyabout the hole is preferably smooth where a seal is to be made duringion transport measurement methods.

Once a plastic chip has been provided with one or more appropriatelyconfigured laser drilled holes, it is then treated with a plasma tomodify the surface. Plasma refers to ionized gases, such as oxygen, air,a combination thereof or other appropriate ionizeable gases. Thecharacteristics, determination and duration of the plasma used can bedetermined using routine experimentation and screened using iontransport measurement methods described herein or in the applicationsincorporated by reference herein.

Generally, the plasma treatment cleans the surface of the material andsurface of the hole. Such cleaning includes removing or reducingconduction substrates and unwanted contaminants. The plasma treatmentpreferably results in a change in charge of the surface and/or surfacesurrounding the hole to charged or more charged, where the preferredcharge is negative, and/or the hydrophilicity to hydrophilic or morehydrophilic. The plasma treatment can also result in the removal orreduction or carbonation and/or conductive surfaces and/or materials.Not wishing to be limited to a particular mechanism, the plasma wouldalso create chemical reactive functional groups on the surface, such asthe surface of the hole. These functional groups can be used in furthermodification of the characteristics of the surface by, for example,further chemical reactions and/or polymerizations to create new oradditional functionalities on the surface, such as the surface of a holeand/or environs thereof. Such modifications preferably would result in amore hydrophilic and/or electronegative characteristic of the surface,more preferably the surface of the hole and surrounding environs on thechip. The resulting functional groups exposed on the surface of thesubstrate would be believed to include, but not be limited to, hydroxylgroups, carboxyl groups, free radicals and the like. The resultingmodified surface would result in a surface that would provide a highquality seal for use in ion transport measurement.

Surfaces can be modified to contain negatively charged groups, includingbut not limited to, sulfate, phosphate, borate, silicate, carboxyl,thiosulfates, thiophosphates, thiocarbonates, borates and the like. Thenegatively charged groups can be provided alone or in combination. Thechoice of chemistry of the modification is within the routine skill ofan artisan and can be made using chemical reactions known in the art.The amount of charge can be varied by altering the negatively chargedgroups, a combination of negatively charged groups, the chemicalreaction used, the duration of the chemical reaction and the like. Otherfunctional groups that can be added, alone or in combination with thenegatively charged groups, include, but are not limited to positivecharge, neutral charge or hydrophobic.

Once a surface has been modified, the resulting chip can be tested forthe ability to form a tight seal in an ion transport measurement. Thedevices and methodologies described herein and in the applicationsincorporated by reference can be used the screen chips to determinepreferable modification methodologies. Generally, it is desired tocreate negatively charged and hydrophilic characteristics of the surfaceof the hole and/or surrounding surfaces is desirable.

The size and shape of the chip can be varied depending on theinstrumentation to be used for ion transport measurement using the chip.Preferably, the chip has a footprint that is appropriate for existingion transport measurement, such as those described herein and in theapplications incorporated by reference. For example, 16 holes arrangedin a linear chip in a linear configuration can be used in these devicesand methodologies. The density of the holes and number of holes can alsobe varies. Preferably, the footprint of the chip would be that of astandard microtiter plate that has, for example, between 9648 and 1536wells, or greater, preferably in multiples of 96. The holes wouldpreferably form a matrix where the holes would generally align wherewells would be in such standard microtiter plates. In that way, existingrobotics and instrumentation could be used to provide reagents andmaterials to locations on a chip.

Another example of the chip and method of making includes a Kapton chipdrilled by laser at about 248 nm, which results in an exit hole the sizeof about 2 micrometers in diameter. The entrance hole would be about 100micrometers in diameter with a thickness of about 150 micrometers. Thedrilled chips are plasma treated for about three minutes. The plasmatreated chips are exposed to a 5% solution of S0₃-triethylamine complexin DMF and shaken for about 30 minutes. The resulting chips are washedwith water and stored in water. The resulting chips are used in iontransport measurement as described herein or in the applicationsincorporated by reference.

Higher throughput ion transport measurement is made possible using chipswith higher hole density and hole numbers. For example, a chip having384 wells can perform 384 simultaneous ion transport measurement assaysas opposed to the relatively small number of holes in existing iontransport measurement chips having between about one and sixteen holes.

Bilayer-Bilayer Junction for Forming Tight Seals and Method of use

Surface modification has come to play an important role in a largenumber of technologies from different disciplines, including adhesion,corrosion inhibition, and chemical separation sciences. The last decadehas witnessed an increasing expansion and interest in supported lipidbilayers as models of biological membranes and as a physiological matrixfor studying a number of phenomena, including membrane-receptor,cell-cell interactions. Another aspect of the present invention is theintroduction of a lipid bilayer, or portion thereof, to produce aninteraction, via a spanning divalent cation, with the lipid bilayer of acell or portion of a cell that is to be the subject of the measurement.Such bilayer-bilayer junction for forming tight seals and method of useare claimed. In this method, a lipid bilayer may be provided to coverthe surface or a portion of the surface of a chip used for ion transportmeasurement. The lipid is preferably attached to a surface of a chip,such as through covalent or non-covalent attachment. Preferably, headgroups of a lipid bilayer are attached to the surface, and a second,inverted layer of lipid is attached via van der Waals forces (ahydrophilic exclusion phenomenon) on top of the first layer such thatthe polar head groups of the second layer are oriented outwards from thesubstrate. The surface thus has a charge that would promote theformation of a tight seal for use in ion transport measurement. Anyappropriate bilayer can be used, preferably having a negatively chargedsurface. In a specific example, one preferable lipid bilayer is onecomprising phosphatidyl-ethanolamine lipid. The lipid head group isattached to the surface of a hole. The surface of the hole can be of anyappropriate material, such as plastic, glass or a combination thereof,such as those described herein, in the applications incorporated byreference, or described in the art. The attachment can be covalent ornon-covalent. Preferably, covalent attachment of lipid head group to theglass surface will be achieved through PE liposome fusions usingmethodologies known in the art. In this configuration the negativelycharged lipid head group moiety, from the lipid outer layer, and thenegatively charged species, of a target membrane, such as a cellmembranes, can be bridged using divalent cations that can coordinatebetween the two polar head units without the necessity for forming acovalent or ionic bond. The resulting interaction between the membranesis expected to form a tight seal for ion transport measurement. The areaof membrane bilayer that is then left bound within the hole on the chipcan be ruptured using methods described herein, in the applicationsincorporated by reference or as known in the art. Examples includepositive pressure, negative pressure and treatment with chemical orbiological agents that can form pores in biological or syntheticbilayers. The resulting structures can be evaluated for the formation oftight seals and efficacy in ion channel activity determinations usingthe methods and devices described herein or in the applicationsincorporated by reference or as known or described in the art.

In another aspect of the present invention, one half of a lipid bilayeris attached to the surface of a chip, preferably in areas in closeproximity to a hole, or aperture, on a chip for use in ion transportmeasurement. The hydrophobic tails of the one half of the lipid bilayerare attached to the chip such that the polar heads are facing outwardson the chips surface such that the polar heads can interact with cellswhen the chip is used for ion transport measurement.

In another aspect of the present invention, only polar heads areattached to the chip. In yet another aspect of the present invention,only charged heads are attached to the chip, and in yet another aspectonly the functional units (which includes only the chemical units thatparticipate in the coordination chemistry that is used to form a seal,via a divalent cation, with the membrane on a cell or particle that isthe subject of the measurement) of the polar heads are attached to thechip. These modified chips can interact with cells to assist in theformation of tight seals for use in ion transport measurement. Thesestructures can be evaluated for the formation of tight seals andefficacy in ion transport measurement using the methods and devicesdescribed herein or in the applications incorporated by reference or asknown or described in the art.

A Method of Making a Pre-Assembled Ion Transport Measurement Cartridgeand Method of use

Another aspect of the present invention is a method of making apre-assembled ion transport measurement cartridge and method of use. Inthis aspect of the present invention, a chip without a hole is providedin a cartridge, such as described herein or in the applicationsincorporated by reference. The assembled cartridge is then oriented forlaser drilling of the chip to form holes for use in ion transportmeasurement. The resulting cartridge with a chip with holes can then betreated for surface modification to promote the formation of tight sealsfor ion transport measurement. The treatment can be any appropriatetreatment; preferably those described herein, the applicationsincorporated by reference herein, or as known or described in the art.These cartridges can be used to perform ion transport measurement asdescribed herein, the applications incorporated by reference herein, oras known or described in the art.

In another aspect of the present invention, a chip without a hole isprovided. The chip can be made of any appropriate material, such asglass, plastic or a combination thereof. The chip can be of anyappropriate thickness, preferably between about 20 microns and about 180microns. The chip is assembled to a cartridge using methods describedherein, the applications incorporated by reference or as known ordescribed in the art. For example, the chip can be attached to thecartridge using adhesives, sonic welding, melting or the like. Thechoice of materials for the chip and cartridge can be readily evaluated,determined and selected depending on the proposed modifications of thestructure, if any. For example, if the structure is to be treated withacid, base, solvents, reactants or a combination thereof, then thematerials should be resistant to degradation or contamination by thesechemicals. Preferable materials for the cartridge include, but are notlimited to plastics of any sort, preferably cyclo-olefins, acrylics,imides, styrenes, and/or copolymers thereof. Likewise, if the structureis to be treated by heat, cold or irradiation, then the structure shouldbe resistant to degradation by these conditions. The chip-cartridgecombination would form one or more wells. Preferably, the chip forms thebottom of a well and the cartridge forms the remainder of the wellitself. Such structures are described herein and in the applicationsincorporated by reference herein.

The resulting chip-cartridge combination is provided on a laser drillingplatform. The chip-cartridge can be provided in any configuration,preferably chip side up or chip side down, more preferably chip sidefacing the laser drill. The laser drilling apparatus would have thecoordinates of the location of the hole to be drilling stored in amemory device that is operated by a central processing unit. Thechip-cartridge combination would be oriented on the drilling apparatususing appropriate structures, such as pins, jigs, flanges, depressionsand the like as is known in the art. The hole to be drilled wouldpreferably be in the center of a well formed by the chip-cartridgecombination, but that need not be the case. The laser drilling apparatuscan be any known or described in the art or commercially available. Suchlaser drilling apparatus can be readily configured and/or programmed forthese operations by the skilled artisan. The laser drilling apparatusdrills holes in the chip in a configuration appropriate for iontransport measurement as described herein, the applications incorporatedby reference or as known or described in the art. Preferably, acounter-bore is made in the chip. Then the hole used to perform iontransport measurement is made. The type, wavelength and energy of thelaser used for drilling is a choice of the artisan based on the type ofmaterial, the thickness of the chip, and the desired characteristics ofthe hole.

The drilled chip-cartridge combination can then be treated to enhancethe formation of tight seals for use in ion transport measurement. Suchmodifications can be any known in the art, but are preferably thosewhich make the chip, the hole and/or the surrounding environs moreelectronegative, electronegative, more hydrophilic, hydrophilic or acombination thereof. Preferable methods of modification are describedherein or in the applications incorporated by reference herein.

The untreated or treated drilled chip-cartridge combination can be usedin ion transport measurement using the methods and devices describedherein, in the applications incorporated by reference herein or as knownor described in the art. The effectiveness of the chip-cartridgecombination, with or without treatment, and the assembly and drillingprocess can be evaluated based on the results obtained and byinspection. For example, the characteristics of the hole can beevaluated using optical and/or electron microscopy. The appropriatenessof the materials for the chip and/or cartridge can be evaluated byvisual inspection, physical testing such as physical stress testing andthe like. Effectiveness of treatment procedures and overall performanceof the unit can be evaluated using ion channel detection methodsdescribed herein or in the applications incorporated by referenceherein.

In another aspect of the present invention, the cartridge is made ofpolysulfone attached to a glass plate having counter-bore holes having adiameter between about 50 micrometers and about 100 micrometers indiameter. The counter-bores would be drilled by a laser of about 193 nmwavelength until about 15 to about 30 micrometers of glass remains. Asecondary hole is drilled to penetrate through to the other side of thesubstrate, with an exit hole the size of about 2 micrometers in diameterwith an entry hole of about 7 micrometers in diameter. The chips arethen treated to increase tight seal during ion transport measurement,such as by treatment with base. The structure is then used in iontransport measurement using the methods and/or devices described herein,in the applications incorporated by reference or as known or describedin the art. The manner by which the cartridge is employed on themeasurement device is either with cartridge side up wherein thecartridge itself forms the upper chamber to which particles are added tobecome the subject of the measurements, or with cartridge side downwherein the cartridge forms the bottom chamber that may form a seal withthe measurement device to provide isolated bottom chambers during themeasurements.

A Method of Layering a Plastic Chip with Glass and Method of use

Another aspect of the present invention is method of layering a plasticchip with a thin sheet of glass and method of use. Generally, a plasticchip is layered with glass wherein the plastic portion forms a supportstructure to maintain the integrity of the thin glass sheet. The glasslayered chip is laser drilled to form holes useful for determination ofion channel activity.

In this aspect of the present invention, a plastic material used to forma chip for ion transport measurement is provided. The plastic materialcan be any appropriate, to including but not limited to polyimide (suchas but not limited to Kapton), polyolefin, cyclo-olefin, or copolymersthereof (such as but not limited to Zeonor), polyacrylate, siliconcontaining polymer, wax (eg, paraffin wax), Parafilm, fatty acid basedpolymers or the like and combinations thereof or copolymers thereof. Theplastic materials are generally characterized as being capable ofproviding a rigid support structure to prevent the fracturing of thelayered glass substrate. The glass substrate is characterized as being10 and 100 μm in thickness.

The plastic material is layered with glass by gluing, by proximityadhesion, or by using methods known in the art. For example, the glassmay be bound to the plastic substrate by UV-curable adhesives, bypressure sensitive adhesives, or by thermal adhesives. Further, a sheetof thin glass may comprise one sheet that covers multiple recordingholes, or may be cut or diced to provide only sufficient glass to covera single recording hole. Holes can be drilled through the glass-layeredplastic using a variety of methods. Preferably laser drilling usingmethods described herein, in the applications incorporated by referenceherein or as known or described in the art can be used. The choice oflaser type, strength, wavelength and duration are the choice of theskilled artisan based on the materials and the nature of the resultinghole desired. Preferably, the surface surrounding or partiallysurrounding the hole is smooth where cells are to interact and iontransport measurements are to be made. Characteristics and dimensions ofsuch holes, and optional counter-bores are described herein, in theapplications incorporated by reference and as known or described in theart.

The holes can be drilled into or thorough the glass layered plasticbefore or after the glass layered plastic chip is attached to acartridge. When attached to a cartridge, the glass layered chippreferably forms at least a portion of the bottom of a well while thecartridge forms the remainder of the well, but that need not be thecase.

The chips can be treated to increase tight seals when used in iontransport measurement procedures. For example, the chips can be treatedwith acid, base, oxygen plasma or other plasma, reactive groups, aloneor in combination. Generally, such modifications result in a chip, holeand/or hole surface that is electronegative, more electronegative,hydrophilic, more hydrophilic, or a combination thereof. Methods ofmodification are described herein, in the applications incorporated byreference and as known or described in the art. Chips can be treatedbefore or after assembly with a cartridge, if such assembly takes place.

The physical nature of the holes and chips, with and without cartridgestructures, can be determined by a variety of procedures. Visualobservation of the holes using light or electron microscopy can be usedto evaluate the dimensions, quality and physical characteristics of thehole and surrounding environs. Stress testing can be used to determinefragility of the structure, such as by dropping a structure from apredefined or variety of heights or subjecting to a predetermined orvariety of pressure measured, for example, in pounds per square inch.

The ability of the chip to perform ion transport measurements,preferably by forming tights seals can be evaluated. The chip, eitheralone or in combination with a cartridge, is used to perform iontransport measurement as set forth herein, in the applicationsincorporated by reference or as known or described in the art.Preferable procedures are readily determined by this method.

A Method of Protecting Fragile Devices and Parts by Packing and Shippingin a Fluid such as Water

Another aspect of the present invention is a method of protectingfragile devices and parts by packing and shipping in a fluid such aswater. Many of the chips, either alone or in combination with cartridgestructures, can be fragile and difficult to transport without breakage.By packaging, storing and shipping fragile structures in a fluid such aswater, breakage can be reduced.

Small, fragile devices and parts are routinely used in a variety ofindustries, including the biological, semiconductor, aerospace, defenseand the like. In the biological industry, a variety of fragile devicesand parts are made of glass. Such fragile devices and parts tend to bedifficult to make and the outsourcing of their manufacture is desirable.For example pipette tips using in ion transport measurement are quitefragile. Also, the chips of the present invention, with or withoutcartridge structures, can be fragile. In order to allow outsourcing ofmanufacture of these fragile structures, it is desirable to have methodsof storage and/or packaging that would reduce breakage during storage atthe manufacture site, shipping, and/or storage at the site of receiptfrom shipping or some other location. The reduction in breakage ordamage to such fragile structures would result in cost savings to themanufactures, recipients and users of such structures.

In order to reduce damage of such fragile structures, the presentinvention provides such fragile structures in a fluid environment withina container. The fluid environment and container are chosen such thatthe fluid does not appreciably degrade the container or the fragilestructure. Preferably, the fluid environment does not cause elementsfrom the container and/or fragile structure to leach out. The fluidenvironment can completely or incompletely fill the container. The fluidenvironment can be a single or multiple fluids. The fluid environmentcan be a single or multiple phases. Multiple phases would include anaqueous and hydrophilic fluid. The fluid environment can be of anyviscosity. The fluid environment is preferably liquid at temperaturesused for storage and shipping. Preferable fluid environments includewater, alcohol, oils and the like.

A Method of Laser Beam Splitting and Method of use to Laser Drill Holesin Ion Channel Chips

Another aspect of the present invention is a method laser beam splittingand method of use to laser drill holes in ion channel chips. The methodmay or may not require a homogenizer to obtain a “top hat” power profilefrom a laser beam source. The laser beam is then optionally masked atthis point in order to provide one or more profiles for laser drilling asubstrate for use as an ion channel detection structure. The laser beamis then passed through a beam splitter to make two or more laserbeamlets. The laser beamlets are then optionally masked to provide oneor more profiles for laser drilling a substrate for use as an ionchannel detection structure. Either the first, second or both of themasking steps can be used. The laser beamlets are then focused through asingle or multiple lenses onto a work-piece. The work-piece is in one ormore parts and is laser drilled to form structures for use in ionchannel detection methods.

The material to be laser drilled can be any appropriate material.Preferred materials include, but are not limited to, glass, ceramics,plastic, silicon, or a combination thereof. Preferred materials arediscussed herein, in the applications incorporated by reference and asknown and described in the art. The selection of a laser—material pairhas been discussed herein, in the applications incorporated by referenceand as known and disclosed in the art.

The laser used in these methods can be any appropriate for the materialto be laser drilled. The selection of a laser and material combinationis within the skill of the artisan and can be determined using methodsdescribed herein, in the applications incorporated by reference and whatis known and described in the art. Preferably, excimer laser processingis used. Excimer laser processing can be useful for projection masktechnologies to machine small features on a parallel scale. Threecomponents of excimer laser processing are the laser, optics and mask.

The laser can be any appropriate laser. A variety of excimer lasers arecommercially available, including but not limited to 351 nm, 308 nm, 248nm, 193 nm and 157 nm wavelength lasers. The choice of wavelengthdepends on the factors of the material to be ablated and the material'setching behavior at that wavelength. Most organic materials ablate atalmost all of the excimer wavelengths, materials such as glass, ceramicsand silicon can provide better results at lower wavelengths. The choiceof laser wavelength can also depend on the damage threshold of opticalcomponents, such as lenses, grids, mirrors and masks, at a givenwavelength.

A preferable optical design for excimer laser ablation is a 5× or 4×reduction ratio optical train. However, other optical designs can beused in the present methods, such as one which incorporates a 20× to 35×reduction ratio. The optical system includes one or more of thefollowing: beam shaping module, beam homogenizer, grids, mirrors,projection lens, and mask. Factors to consider for designing anappropriate optical train design include the damage threshold ofcomponents at a given wavelength of laser, fluence required forablation, area to be ablated, feature size to be ablated and therepetition rate.

The mask can be made of any appropriate material. Preferred materialsinclude, but are not limited to, chrome on quartz, aluminum on quartz,dielectric on quarts and metals. Preferred metals include, but are notlimited to, stainless steel and molybdenum. The choice of materials formasks includes a consideration of the process fluence and repetitionrate of laser. Masks should be chosen to withstand the thermal loadgenerated by laser irradiation during processing cycles. The opticstrain design should be chosen such that laser irradiation at the maskplane is lower than the mask damage threshold.

Laser ablation of holes in a substrate to form structures for iontransport measurement can take a variety of shapes. Preferred shapes areconical, characterized by a plurality of counter-bores and a final holethrough the substrate. The following dimensions are provided by way ofexample and are not intended to be limiting. The first, and largest,counter-bore is preferably about 100 micrometers in diameter at itsopening and about 100 micrometers deep. A second counter-bore ispreferably about 60 micrometers in diameter at its opening and about 40micrometers deep. The last hole, which passes through the structure, ispreferably about 7 micrometers in diameter at its widest opening, about20 micrometers in depth, and emerges from the piece at about 1.8micrometers to about 2.0 micrometers in diameter at is smallest opening.The total depth of the hole is about 150 to about 160 micrometers deepand made of glass assuming that all holes are drilled from the sameside, which need not be the case. For the three holes, two counter-boresand one through hole, operating parameters follow: Fluence (mJ/cm2)range from about 3,000 to about 4,000. Repetition Rate (Hz) range fromabout 200 to about 400. Number of pulses range from about 150 to about800. Etch depth (micrometers) range from about 10 to about 140. Etchrate (micromters/pulse, range from about 0.05 to about 0.15.

For example, as shown in FIG. 50, FIG. 51, and FIG. 52, a variety ofchip configurations can be made using this technology. As shown in FIG.50, a 16×24 array of holes or a 16×1 array can be made using a singledrill time. Alternatively, as shown in FIG. 51, four holes can bedrilled at a time to form any number of holes, including the 16×24 arrayof FIG. 50. As shown in FIG. 52, nine holes can be drilled at a time toform any number of holes, including the 16×24 array of FIG. 50.Alternately, two, three, or six holes may be drilled at one time by thesame approach. A large objective lens can be used to make a large numberof holes in a chip. Alternatively, multiple laser beamlets may bedirected to multiple parts to fabricate multiple parts at a time, usingregular objective lenses for each beamlet.

The chips made using this method can be tested for ion channel detectionusing methods described herein, in the applications incorporated byreference and as known and disclosed in the art. The quality andcharacter of the holes can be evaluated visually, such as by lightand/or electron microscopy, to evaluate the structures provided by thesemethods.

A Method of Making Glass more Readily Wet Etched, Ion Channel Chips madeusing this Method, and Methods of use Thereof

Another aspect of the present invention is a method of making glass morereadily wet etched, ion channel chips made using this method, andmethods of use thereof. Generally, the invention converts glass to aform that is more readily wet etched by interaction with a laser in theUV range. The glass that is exposed to such laser light becomes modifiedand is more readily wet etched. The methods are preferably used formaking counter-bores, glass thinning and through-holes. Glassappropriate for such procedures is an amorphous glass or glass ceramicthat it thermal sensitive. The methods can also make the glass morebiologically compatible. An example of such glass is commerciallyavailable from Invenions or Schott, and has a product name of Foturan.

In one example, a piece of Foturan ceramic glass about 160 micrometersthick is exposed to UV laser light with a diameter of about 100micrometers. The glass is then wet etched and the resulting hole orcounter-bore evaluated via light or electron microscopy. Depending onthe energy of the laser and the duration of exposure, the laser does notneed to appreciably ablate the glass and therefore the lower energyrequirements for the laser allow a single exposure to cover a largerarea that may be the size of the entire substrate at once, which througha projection mask may be used to expose all of the counter bore areas atonce on a chip. Upon wet etching, however, a hole or counter-bore isprovided. By altering the characteristics of the laser and exposuretimes, and by altering the characteristics of the wet etching,counter-bores and/or through holes can be made through glass. The sizeand shape of the counter-bores and through-holes preferably havecharacteristics of size and shape as described herein, but that need notbe the case. The resulting holes can be treated as described herein orin applications incorporated by reference herein. The treated anduntreated chips can be tested for effectiveness in ion channel detectionmethods using methods described herein an applications incorporated byreference.

A Method for Bonding Glass to Glass and a Products Produced by thisMethod

Another aspect of the present invention is a method for bonding glass toglass and the products produced by this method. The method makes use offlat surfaces of glass that an operator desires to bond together. Thesurfaces of the two pieces of glass that are to be bonded can beuntreated or treated independently with acid and/or base, then broughtin close apposition to one another to produce a laminate of the twopieces. The resulting laminate is heated to bond the two pieces of glasstogether. Alternatively, the two pieces of glass can be untreated ortreated independently with acid and/or base. A thin layer of Na₂SiO₃powder or solution is placed between the two pieces of glass that arebrought in close apposition to one another. The laminate is heated tomelt the NaSiO₃, which requires a lower temperature to melt, and thusbond the two pieces of glass together. The invention includes productsproduced by this method.

In one preferred aspect of the invention, a first surface of glass istreated with acid. A second surface on another piece of glass is treatedwith base. The acid treated surface is placed in close apposition orcontact with the base treated surface. The laminate is heated such thatthe first piece of glass and second piece of glass are bonded together.

In another preferred aspect of the invention, a first piece of glass maybe treated with base. A second piece of glass may be treated with base.The first surface is placed in close apposition or contact with thesecond surface after Na₂SiO₃ powder or solution has been layered betweenthem. The laminate is heated such that the first piece of glass andsecond piece of glass are bonded together.

The resulting bonded glass can be used for a variety of purposes,including bonding glass with multiple large holes (for example, 384holes through a 2 or more mm thick glass) to a thinner piece of glassintended to be laser drilled to form structures for use in ion channeldetection methods. Such laser drilled structures can be used in themethods and devices of the present invention to detect ion channelactivities. Such methods are described herein and in the applicationsincorporated by reference.

An In Situ Method of Making a Cartridge for use in Ion TransportMeasurement and Methods of use.

Another aspect of the present invention is an in situ method of making acartridge for use in ion transport measurement and methods of use. Achip with at least one hole for use in ion transport measurement isprovided to an instrument used to make ion transport measurement. Thechip is placed such that gaskets are provided on the top surface of thechip and the bottom surface of the chip to form top and bottom chambers.The chambers leave the hole or holes exposed for use in ion transportmeasurement. A cross section of such a configuration wherein the gasketsengage a chip with holes is provided in FIG. 53.

In this aspect of the present invention, a bare chip of the presentinvention for use in ion transport measurement is provided without acartridge or with a cartridge that comprises only a frame intended toprovide edge support for the fragile substrate. The cartridge is formedin situ within an instrument for use in ion transport measurement. Thecartridge is formed by gaskets within the instrument that result inupper chambers and lower chambers for use in methods of ion transportmeasurement. In this way, a completed cartridge need not be provided.The gaskets can be cleaned and reused with a series of chips.

The chip can be any chip with one or more holes for use in ion transportmeasurement described herein, in the applications incorporated byreference or known or described in the art. The holes can be provided inany appropriate configuration, but are preferably in a linear or matrixarrangement that corresponds with standard multiwell plateconfigurations that are commonly available on the market, and for whichstandard robotics interfaces are available to interface with the chip.

The gaskets can be of any appropriate material that can form awater-tight or water resistant seal with the chip. Preferable materialsinclude, but are not limited to, rubbers, plastics, silicone, gels andthe like.

In operation, a chip is provided and inserted into an instrument usedfor ion transport measurement. The chip is oriented such that when thegaskets engage the chip, the holes remain exposed and upper and lowerchambers are formed. The gaskets engage the chip to form such chambers.The upper and lower chambers have electrodes for use in ion transportmeasurement. Appropriate reagents are provided to the upper and lowerchambers for ion transport measurement. Cells are provided to onechamber, preferably the upper chamber, and a cell engages the hole toform a tight seal. Methods described herein and the applicationsincorporated by reference to assist in forming a tight seal. Forexample, negative and/or positive pressure in either chamber, andpreferably in the bottom chamber, can be used to encourage a cell toform a tight seal with the hole. Ion transport measurement is thenperformed.

After ion transport measurements are performed, the reagents and chipare removed and the gaskets cleaned and dried. Another chip is theninserted into the instrument and the process is repeated. The processcan be automated such that an operator of the instrument need not handlethe chips during engagement and/or disengagement with the gaskets.

A Vacuum Gasket for use in Ion Transport Measurement and Methods of useThereof

Another aspect of the present invention is a vacuum gasket for use inion transport measurement and methods of use thereof. One aspect of thepresent invention is depicted in FIG. 54. A gasket is provided to engagea chip for use in ion transport measurement. The gasket can be made ofany appropriate material that can form a water tight or water resistantseal with a chip. Preferred materials include, but are not limited to,plastics, rubbers, silicones, gels and the like. The gasket includes avariety of structures to engage a chip and form chambers for use in iontransport measurement. These structures include 0-rings, vacuum holesand vents.

O-rings are provided at locations to correspond to holes on chips foruse in ion transport measurement. The O-rings can be provided in anyappropriate configurations, but are preferably in a linear or matrixconfiguration. The O-rings are used to engage a chip and form bottomchambers for use in ion transport measurement. The shape of the O-ringsmay be round and contain a conduit connection to a pressure controllerapparatus, or the may be elongated to allow connection to two conduitsthat may be used for both pressure control and for introduction andexchange of ion transport measurement fluid into the bottom chambers.

Vacuum holes are provided such that negative pressure can be applied tothe chip to assist in maintaining a firm seal between the chip and thegasket. The vacuum holes are located within the interstitial spaces notbound byoutside the O-rings to produce a vacuum chamber, in the spacebetween the gasket and the chip, designed to attract and retain the chipon the gasket, and at the same time to produce the appropriate amount ofcompression of the O-rings to ensure airtight seals are formed betweenthe O-rings and the chip surface such as to seal off the inside of theO-rings as independent and electrically isolated chambers that are ableto provide pressure control for the engagement of cells or otherparticles having ion transport. The vacuum can also reduce or preventcross-talk between wells during ion transport measurement by preventingaccumulation of fluids or reducing exchange of fluids or electricalsignals between wells. The amount of negative pressure to be used can bereadily determined using routine experimentation based on a variety offactors, such as the strength of the materials used to make the chip andthe quality of the firm seal between the gasket and chip.

Vents are optionally provided to allow inflow of air into theinterstitial spaces formed when a chip engages the gasket. The inflow ofair when negative pressure is applied can be used to regulate thequality and character of the firm seal between the chip and gasket. Theinflow of air also allows the interstitial spaces to be cleared ofexcess or leaked fluids from the chambers and/or wells. Ridges areoptionally provided along the outside edge or perimeter of gasket. Theridge or ridges are used to assist in having the gasket engage the chip.

Upper chambers for use in ion transport measurement may be provided onthe chip itself, or can be provided by the instrumentation as disclosedherein, in the applications incorporated by reference or as known ordisclosed in the art.

In operation, a gasket is provided, optionally within an instrument foruse in ion transport measurement. A chip for use in ion transportmeasurement is provided and engaged with the gasket. The chip engageswith the gasket such that holes in the chip align with the o-ringsprovided on the gasket. In that way, chambers, and preferably lowerchambers, are formed for use in ion transport measurement. The lowerchamber includes electrodes for use in ion transport measurement. Uppergaskets are provided on the chip or the instrumentation. Upper chambersinclude electrodes for use in ion transport measurement.

When the assembly is completed, negative pressure is provided throughthe vacuum hole to form a firm seal between the chip and the gasket. Theo-ring structure results in the formation of interstitial spaces due tothe elevation of the o-ring structure as set forth in FIG. 54(B) as “d”.

Reagents are provided in the upper and lower chambers for ion transportmeasurement. Cells are added to the upper or lower chamber, preferablythe upper chamber. Cells are encouraged to engage the hole on the chipto form a tight seal as described herein, the applications incorporatedby reference and as known or described in the art. For example, positiveand/or negative pressure, applied to either chamber independently of thevacuum applied to the interstitial space, can be used to encourage acell to engage a hole. Ion transport measurements are then performed.Upon completion of such determinations, the chip is removed from thegasket, a new chip is provided, and the process is repeated.

A System for Automated Processing of Chips for use in Ion TransportMeasurement

Another aspect of the invention is provided in FIG. 55. Briefly, a chipis provided in a treatment solution. A treated chip is picked up by astructure having negative pressure to engage a chip. The chip is passedover structures providing negative and positive pressure to dry thechip. The chip is then further dried in a structure having negativepressure and optionally moving the chip from side to side. The chip isthen removed using the structure having negative pressure and assembledinto a cartridge, a storage structure or an instrument for use in iontransport measurement.

A structure having negative pressure in order to engage and move a chipis provided in FIG. 55(A). Negative pressure is provided to a manifoldhaving a plurality of projections all in operable communication with asource for negative pressure. The negative pressure is provided at alevel that is sufficient to engage a chip but not damage a chip and canbe determined using routine experimentation. The chip engages theprojections preferably in a configuration such that the holes in thechip are not engaged by the projections. A chip can be released byreducing or stopping the negative pressure.

The structure of FIG. 55(A) can be used to transport chips for anypurpose. One use of such a structure is depicted in FIG. 55(B). Thestructure of FIG. 55(A) can be used in one or more of the chip transportsteps depicted in FIG. 55(B). A chip is provided and is placed in atreating solution. Once treatment is completed, the chip is passed neara negative pressure source and a positive pressure source to dry thechip. The chip is then placed in a drying structure having negativepressure to further dry the chip of any solution that may be present ontop of the chip and seep around to the bottom side. The chip can beplaced in a holding structure such as described herein, in applicationsincorporated by reference or known or described in the art. The chip canbe moved in any direction in order to promote drying of the chip. Thechip is then transported to another location for packaging, storage, useor further processing. Further processing includes incorporation into acartridge as described herein, in applications incorporated by referenceor as known or described in the art. In one aspect of the presentinvention, the chip is provided to an instrument for use in iontransport measurement as described herein, in the applicationsincorporated by reference or as known or described in the art.

A Method for Making a Silicon-Based Chip with Laser-Drilled Holes, andSurface Modifications

In another preferred embodiment, holes can be drilled into a siliconwafer using a green laser. A copper vapor laser (CPL) that produceswavelengths of 510.6 nm (green) and 578.2 nm (yellow) at an intensityratio of about 2:1 can be double-staged to produce 255 nm and 289 nm UVpower. The CVL produces high power (about 10W) at 10-20 kHz. After laserdrilling of the holes, the surface of the wafer can be oxidized toproduce a SiO₂ surface.

A Device that can Hold Chips for Methods of Treating Chips and/or forStorage of Chips

Another aspect of the present invention is provided in FIG. 56. Thestructure has a hinged top structure that engages a bottom structure,wherein the top structure and bottom structure are reversibly engagedusing a clip. The bottom structure includes separate structures to holdor otherwise engage chips of the present invention at a location forchips. In the structure set forth in FIG. 56, the location for chip isconfigured for long thin chips. Other configurations for other chipsizes can be readily designed and made within the scope and spirit ofthe present invention. In operation, the top structure is lifted awayfrom the bottom structure via the hinge. One or more chips are placedindividually in the location for chips. Preferably, only one chip isplaced in one location for chip. The top structure engages the bottomstructure and is reversibly held in place by the clip. When loaded withchips and clipped in the closed position, a small gap space is allowedfor free movement and wetting of the chips within a fluid environment.Further, the design of the structure allows complete wetting of allareas of all chips while held within a fluid environment. The structurewith the chips can be used to store chips or be used to hold chipsduring treatment or various steps of fabrication. Alternatively, thestructure with chips can be provided to instrumentation and/or roboticsfor movement of chips, such as during assembly. The structure can bemade of any appropriate material, such as but not limited to plastic,glass, rubber and the like. The structure is preferably made of plasticand is preferably made using injection molding.

The aspects of the invention disclosed herein can be combined to makenew embodiments that are also within the scope of the invention. Theaspects of the invention disclosed herein, such as, but not limited tochip designs, chamber designs, electrode arrangements and connections,through-hole designs and manufacture, fluidics arrangements, etc. can becombined with other features described herein, known in the art, orfeatures that are developed in the future.

The following applications are incorporated herein by reference in theirentireties: U.S. patent application Ser. No. 10/428,565, filed May 2,2003; U.S. patent application Ser. No. 10/351,019, filed Jan. 23, 2003;U.S. patent application No. 60/380,007, filed May 4, 2002; PCTapplication PCT/US03/14000, filed May 2, 2002; U.S. patent applicationSer. No. 10/104,300, filed Mar. 22, 2002 entitled “Biochips IncludingIon Transport Detecting Structures and Methods of Use” naming Wang etal. as inventors; U.S. patent application No. 60/351,849 filed Jan. 24,2002 entitled “Biochips Including Ion Transport Detecting Structures andMethods of Use” naming Wang et al. as inventors; U.S. patent applicationNo. 60/311,327 filed Aug. 10, 2001, entitled “Biochips Including IonTransport Detecting Structures and Methods of Use” naming Wang et al. asinventors; U.S. patent application No. 60/278,308 filed Mar. 24, 2001,entitled “Biochips Including Ion Transport Detecting Structures andMethods of Use” naming Wang et al. as inventors; PCT applicationPCT/US02/11161, entitled “”, filed Mar. 22, 2001, naming as inventors;U.S. application Ser. No. 10/642,014 filed Aug. 16, 2003; and U.S.provisional patent application No. 60/474,508 entitled “Biochip Devicesfor Ion Transport Measurement, Methods of Manufacture, and Methods ofUse” filed May 31, 2003, naming Xu et al. as inventors.

All headings are for the convenience of the reader and should not beused to limit the meaning of the text that follows the heading, unlessso specified.

EXAMPLES Example 1 Device for Ion Transport Measurement Comprising UpperChamber Piece and Biochip

Upper chamber pieces with 16 wells having dimensions of 84.8 mm(long)×14mm(wide)×7 mm(high) were injection molded with polycarbonate or NORYL®material. The distance between centers of two adjacent wells was 4.5 mm.The well wall was slanted by 16 degrees on one side and 23 degrees andcontoured on the other side to allow guidance for cell delivery. Thewell holes had a diameter of 2 mm.

A biochip with 16 laser-drilled recording apertures had dimensions of 82mm (long)×4.3 mm (wide)×155 microns (thick). The distance between thefirst hole and a narrow edge is 7.25 mm. The holes were laser drilled tohave two counterbores of 100 microns (diameter)×100 microns (deep) and25 microns (diameter)×35 microns (deep), respectively. A final throughhole was drilled from the side of the counterbores and had a 7 to 9micron entrance hole and a 2.0 micron exit hole with a total throughhole depth of 20 microns. Chemical treatment with acid and base was doneas described in Example 3.

The treated chip was attached to the upper chamber using UV epoxy glue.

Devices produced using this methods had na Re of ˜2 MOhm with standardES and IS solutions, and an average Ra of ˜6.0 MOhm using RBL cells witha standard pressure protocol described herein.

Example 2 A 52-Chip Bench Mark Study

We have conducted a bench mark study using 52 single-hole biochipstested using a CHO cell line expressing the Kv1.1 potassium channel. Theresult demonstrated a 75% success rate as determined by the followingcriteria: 1) achievement of sealing of at least one gigaohm (a“gigaseal”) within five minutes of cell landing on a hole, and 2)maintenance of Ra of less than 15 MOhm, and Rm of greater than 200 MOhmthroughout 15 minutes of whole cell access time.

Chip Fabrication

Patch clamp chips were designed at Aviva Biosciences and fabricatedusing a laser-based technology (without an on-line laser measurementdevice). The K-type chips were made from ˜150 micron thick cover glass.The ion transport measuring hole structures had ˜140 micron doublecounterbores and final through-holes of ˜16.5±2 micron depth. Theapertures on the recording surface had a diameter of 1.8±0.5 microns.The recording surface was further smoothed (polished) by laser.

Surface Treatment

Chips were received from FedEx overnight service and were inspected forintegrity and cleanness. About 5% of the chips were excluded fromfurther treatment in this process. Selected chips were then treatedaccording to Example 3. Treated chips were stored in ddH₂O for 12 to 84hours before the tests.

Batch QC for Chips

Chips were acid and base treated in batches of 20˜25. Four to six piecesof each batch were randomly picked for testing their patch clampperformance with CHO-Kv1.1 cells in terms of speed to seal and stabilityof the whole cell access. Batches with <75% success rate were excludedfor the 50-chip tests.

Cell Passage

CHO-Kv1.1 cells (CHO cells expressing the Kv1.1 ion channel) betweenpassage 47 and 54 were split daily at 1:10 or 1:15 for next-dayexperiments. Complete Iscove media (Gibco 21056-023) with 10% FCS,1×P/S, 1×NEAA, 1×Gln, 1×HT with 0.5 mg/ml Geneticin was present in mediaused to passage cells and not present in media used to grow cells fornext-day experiments.

Cell Preparation

Cells were isolated using the protocol for CHO cell preparationdescribed in Example 6. After isolation, cells were resuspended in PBScomplete media and passed through a 20 micron polyester filter into anultra-low cluster plate (Costar 3473). The cells were used for the studybetween 30 minutes and 3 hour 30 minutes after the filtration.

Cell QC

Isolated cells were QC'ed with conventional pipette patch clamprecordings for their speed to seal, break-in pressure, and Rm and Rastability. Freshly pulled pipettes were typically used within 3 hrs.Only cell preparations that passed the pipette QC were used for the50-cell tests. About 50% of the preparations out of approximately 30cell isolations passed and were used for this study.

Solutions

Intracellular solution was made according to the following formula: 8 mMNaCl; 20 mM KCl; 1 mM MgCl2; 10 mM HEPES-Na; 110 mM K-Glt; 10 mM EGTA; 4mM ATP-Mg; pH 7.25 (1M KOH3); 285 mOsm.

Aliquoted at 10 ml per 15 ml coming centrifuge tube, and stored at 4° C.

Extracellular solution (PBS complete) was DPBS (1×), with glucose,calcium and magnesium (Gibco cat# 14287-080).

This solution contained:

0.9 mM CaCl2, 2.67 mM KCl, 1.47 mM KH2PO4, 0.5 mM MgCl2, 138 mM NaCl,8.1 mM Na2HPO4, 5.6 mM Glucose, 0.33 mM Na-pyruvate, pH 7.2-7.3, 295mOsm.

Chip QC (Quality Control)

For each recording, the chip was assembled into a two-piece cartridge,and the lower and upper chambers were filled with intracellular andextracellular solutions, respectively. The chip was further QC'ed byinspection under the microscope and seal-test resistance measurement.Chips that showed a dirty surface, visible cracks and/or had a sealtestresistance greater than 2.1 MOhm were excluded.

Experiment Settings

Chips that passed QC underwent electrode offset and the overallrecordings were done with 4 KHz bass filter. Cell landing was monitoredon computer screen.

Criteria

A simple description of a positive result is: chips that achievedgigaseals and gave Ra<15 MOhm and Rm>200 MOhm throughout 15 minrecording period.

Results

A total of 58 chips were tested, 6 of which were excluded from finalanalysis. Out of the 52 cells included, 39 (75%) passed the testcriteria. 43 (83%) achieved at least 12 minutes of continuous highquality recordings (Ra<15 MOhm; Rm>200 MOhm); 47 (90%) achievedgigaseals.

Success Rate

Success duration is plotted in FIG. 24 a. Accumulative success rate isplotted in FIG. 24B. Success rate was consistent throughout the tests,which suggests that most of the critical experimental parameters wereunder control. 75% is a representative success rate under the currentcontrolled conditions.

Electrode Resistance (Re)

90% of the electrodes selected for the tests had Re between 1.3 to 2.0MOhms (FIG. 25). A total of 81 chips were mounted and tested. 23(28%)failed the QC test, among which 15(18.5%) were due to Re>2.1 MOhms.5(6%) chips were screened out because of their dirtiness of surface;3(4%) had blocked or cracked holes. Chips were not screened at low Revalues. The reason behind the 2.1 MOhm cut off is that historicallychips with the current geometry (double counterbore) showed lower than75% success rate in achieving the test criteria. Re is more or lessnormally distributed except for a slightly higher peak at ˜1.3 MOhm.

Break-In Pressure

Break-in Pressure is an important parameter for cell condition. Duringthe tests, break-in pressures were tightly distributed between −100 to−130 Torr (FIG. 25). Our previous findings suggest that seals with morenegative break-in pressure are likely to have higher and unstable Ra,while seals with lower break-in pressure are likely to have lower andunstable Rm.

Membrane Resistance (Rm)

After break-in, Rm was mostly between 0.5 to 2 MOhm (FIG. 26). Ending Rmhad a similar distribution, but more skewed to lower values. This isconsistent with the deterioration of Rm over time. However, the amountof Rm deterioration was surprisingly small, which suggests that theseals were very stable during the 15 minutes test periods.

Access Resistance (Ra)

Initial Ra had a normal distribution centered at 7 MOhm. 80% of theseals had Ra starting from below 10 MOhm. In most cases, Ra increasedduring the 15 minutes with an ending value near 11˜13 MOhm. In order tominimize disruption of the seals, great effort was not made trying tomaintain minimal possible Ra. It is not known what the ending Ra wouldbe and what percentage of seals would lose Rm if such efforts were made.

Typical Recordings

FIG. 27 shows the recordings from a typical experiment. The top panelshows recordings right after break-in, while the bottom panel showsrecordings at the end of 15 minutes. The middle graph plots the Rm andRa values over the 15-minute period. There is no observable decrease incurrent in most of the cells recorded. The decrease in Rm and increasein Ra are typical. In addition, Rm and Ra typically plateau out after˜10 minutes to steady levels. We recorded the last cell (#58) for anextended period of time. The cell lasted for about 30 minutes within theconstraint of our test parameters.

Example 3 Treatment of Ion Transport Measurement Chips to Enhance theirElectrical Sealing Properties

Detailed Procedure: (referenced to step numbers below). All incubationprocesses were carried out in self-made Teflon or Noryl fixturesassembled in a glass tank while shaking (80 rpm, with C24 IncubatorShaker, Edison, N.J., USA). Water was always as fresh as practical froma water purification system (NANOpure Infinity UV/UF with Organic freecartridge). Nitric acid was ACS grade (EM Sciences NX0407-2, 69-70%).Sodium hydroxide was 10 N, meeting APHA requirements (VWR VWR3247-7).When necessary, chips were inspected for QC before and after treatment.The protocol used was:

1. 3 hour shaking incubation in 6M nitric acid at 50 degrees C.

2. 6×2 minute rinses in DI water at room temperature.

3. 60 minute incubation in DI water (shaking)

4. 2 hour shaking incubation in 5M NaOH at 33 degrees C.

5. 6×2 minute rinses in DI water at room temperature.

6. 30 minute incubation in DI water (shaking) at 33 degrees C.

7. Chips were stored in DI water at room temperature. A vial used forstorage was filled to the neck to minimize air space.

Chips treated according to this protocol demonstrated enhancedelectrical sealing when tested in ion transport detection devices.

Example 4 Achieving Seals with Inverted Chips

A biochip was fabricated from Bellco D263 or Coming 211 glass ofthickness of ˜155 micron. The 16 laser-drilled recording apertures onthe chip had dimensions of 82 mm (long)×4.3 mm (wide)×155 microns(thick). The distance between the first hole and a narrow edge is 7.25mm. The apertures were laser drilled to have one counterbore of 100microns (diameter)×125 microns (deep). A final through hole was drilledfrom the side of the counterbores and had a ˜10 micron entrance hole and4.5 micron exit hole with a total through hole depth of 30 microns.After standard chemical treatment as described in Example 3, the biochipwas mounted to an upper chamber piece described in Example 1 in invertedconfiguration such that the counterbore side faced the upper chamberpiece (where RBL cells were added). Recordings were done with a deviceadapted to Nikon microscope as described in Example 5. Typical recordingparameters such as Rm and Ra over time are shown in FIG. 27, The tablein the figure shows the average and standard error of parameter valuesmeasured.

Example 5 A Biochip Device Adapted to a Microscope and havingFlow-Through Lower Chambers

This device is shaped to take advantage of an existing mounting point onthe Nikon microscopes by positioning the device into an aperture withinthe microscope stage. It is round, with an edge intended to prevent itfrom falling through the hole on the stage. The depth of the device isintended to hold the functional portion of the biochips as well as thecells that are added to the biochip at testing time at a convenientfocal point within the focal range of the microscopes, ie: atessentially the same level as the upper platform of the microscopestage.

In this design, a biochip cartridge that has a base-treated glass chipsealed to an upper chamber piece may be assembled onto a microscopestage-mounted lower chamber piece that allows simultaneous or sequentialtesting of all recording apertures while simultaneously observing theexperiment's progression microscopically. The tester consists of ametallic base plate, in this case made of aluminum, shaped to insertonto a microscope stage, and sculpted to support and align a multi-wellperfusion lower chamber piece. A gasket is inserted over the lowerchamber piece, then the cartridge, which is clamped onto the gasket bycompression via a clamp assembly that bolts onto the base plate usingfour thumb-screws. The lower chamber piece is made of plastic andcontains an array of 16 holes for inflow of intracellular solution, andanother 16 holes for outflow of same. The 32 holes emerge on the topsurface of the part in alignment with the recording apertures of thebiochip. The gasket is made of PDMS and situates between the lowerchamber piece and the chip, and contains slits, or holes, that alsoalign between the emerging holes of the perfusion conduits of the lowerchamber piece, and the recording apertures of the chip, such than anintracellular “lower” chamber is formed within the array of slits orholes in the gasket. An electrode of silver-silver chloride isintroduced into each of the 16 holes along one side of the bottomchamber to function as the voltage-clamp electrode. The device is madeup of 1) a metallic base, specifically, but not exclusively, stainlesssteel, 2) a transparent lower chamber piece, made from polycarbonate(but could be any other convenient transparent substance) 3) electrodesinserted into the tubes of the inner chamber array, made from wires ofsilver or any other conductor capable of being used as a voltage sensingand current-delivering electrode, and attached to a connector on theouter side of the inner chamber array, 4) inert tubing glued to thetubes of the inner chamber or any other means that may provide aconnection for a fluid conveyance system, in this case made from glass,5) a gasket that provides a seal between the inner chamber tubing andthe biochip cartridge, where the gasket simultaneously comprises theinner chamber, in this case made of PDMS, 6) a biochip mounted onto thetest apparatus over the gasket, and held in place by a guidance system,in this case pins inserted into the plastic bottom chamber array body insuch a way as to restrict movement of the biochip while simultaneouslyguaranteeing alignment of the biochip's recording surface with the innerchambers, 7) a clamp assembly intended to apply sufficient pressure ontothe biochip cartridge so as to generate a seal between the bottom thechip and the gasket, and 8) an array of electrodes shaped and orientedso as to enter into the top wells of the biochip cartridge, all 16 at atime, and where all electrodes are connected together so as to provide areference electrode in the top-chamber of the cartridge.

1) Metallic Base Plate:

This base plate serves multiple functions. First, the metallic bodyserves as an electrical noise shield for the bottom side of the testchamber. It completes a type of faraday cage that is contiguous with thegrounded stage of the microscope, and with a small cage that must beinstalled around the stage of the microscope if no faraday cage is to bepresent in a wider area around the microscope. Secondly, the metal baseis carved on the top side so as to catch any fluids that may leak orspill and prevent the contamination of the microscope with said fluids.To this end, the base plate is sealed, with silicone glue or withsilicone grease (vacuum grease) or with any other such viscousimmiscible substance (eg: Vaseline) to the transparent inner chamberarray described in (2). Third, the base plate may be shaped to optimizeits use with a particular microscope. Specifically, in our case it wasdesirable for the base plate to be cut to fit onto the 107 mm circularcutout hole of a Nikon microscope. Fourth, the base plate is drilled andtapped so as to provide a mounting point for the inner chamber array andfor the clamp of the tester. Its design is such that it will ultimatelyhold the recording aperture of the cartridge within a few millimeters ofthe level of the top of the microscope stage so as to ensure that thechip function may be monitored within the focal range of the microscope.FIG. 2 illustrates the design of the base plate as adapted for the NikonMicroscope.

2) Transparent Inner Chamber Array:

The lower chamber piece may also be referred to as an inner chamberarray, or an intracellular chamber array. For the convenience of beingable to view under a microscope the progression of an experiment, itshould be made of a transparent material. Polycarbonate was chosen forits ease of machining. It is shaped to support a cartridge over it, andprovide tubing connections along the long edges of either side thecartridge, as well as to provide connections to electrodes placed insideone of each pair of tubes (holes in the material that function as such)supplying each recording aperture of the chip. The tubes drilled intoeach side provide a connection between the edge of the part andsomewhere near the center, then another tube drilled perpendicularlyfrom the top surface to connect to each tube coming from the edge. Theemerging tubes at the top surface are located so as to provide an inflowand an outflow of solution in the bottom wells. No well is containedwithin this part but the bottom wells are instead created by an openingwithin the gasket material. The inflow and outflow holes are separatedfrom one another so as to leave an untouched area of surface that willbe easy to see through with a microscope so as to visualize therecording aperture during experimentation. To this end, the top surfacethat is in opposition to the chip should remain untouched with theexception of the emerging inflow and outflow holes and as well thebottom surface should remain untouched so as to not disrupt transparencyof the part. Each tube (or hole) leading to the edges of the part shouldconsist of a means for interfacing it to external tubing (seedescription of part 4) that provides delivery of solutions, as well aspneumatic pressure control. One of the tubes going to the edge of thepart is left longer so as to house an electrode (wire) that isintroduced into the lumen of the tube. The added length also allows fora second segment to be glued onto the top surface so as to house theconnectors for the electrodes. The top surface of this part is trimmeddown around the periphery of area covered by the cartridge so as toprovide an edge that functions to hold the gasket in place duringmounting and unmounting of the cartridge. Further, between each pair ofinflow and outflow holes for each bottom well is a cut intended toprevent wetting of the gasket material to span from one bottom chamberto adjacent bottom chambers. This part as a whole contains 6 holes 2 mmin diameter to hold 6 pins that function to keep the cartridge alignedduring mounting. It also contains a further 4 holes to hold 4 spring-pincompression contacts that function to provide an electrical connectionfor an early version of the cartridge. The present version of thecartridge does not require these contacts, however they were kept inplace so as to prevent contact with the gasket before the clamp part ispressed down during the mounting. Finally, two more holes are present soas to use two screws to hold the part onto the base plate. FIGS. 4-10illustrates the arrangement of the lower chamber piece.

3) Inner Chamber Electrodes:

Each bottom well segment contains an electrode, which in this case is asilver wire that will be periodically chlorided. The wire is insertedinto the lumen of the longer hole and bent upward into the connectorarray. The segment of wire is sufficiently long that it will remainexposed within the lumen of the longer tube after the inert tubinginterface parts are glued into place, and the other end is soldered to aconnector, in this case an array of 1 mm female pin-connector socketsinserted into holes in the part.

4) Inert Tubing Interface:

Into each hole is inserted an inert tube segment (in this case made fromglass) that is fixed in place with epoxy glue. Epoxy is chosen onlyin-so-much-as it is preferred for bonding glass to polycarbonate. Thetubing segments are sufficiently long to butt against a countersunkensegment of the tube drilled into the inner chamber array and stick outof the part enough to hold a segment of silicone tubing that ispress-fit onto the glass segment. This junction should withstand apressure greater than two atmospheres positive pressure, and greaterthan 700 mmHg vacuum pressure. It was determined that 3 to 5 mminsertion into the silicone tubing was sufficient to accomplish thisrequirement. FIG. 7 illustrates the glass tubing mounted into thepolycarbonate inner chamber array.

5) Gasket:

For convenience the flexible gasket may be molded from curing PDMS. Thegasket contains a raised edge on the bottom side that surrounds thechambers as a whole and is intended to hug an edge present in the sameperiphery on the inner chamber array so as to hold the gasket in place.The gasket has oblong holes in it that align over the exit and entranceholes of the inner chamber array for each chamber of the array. On thetop surface of the gasket is a set of squared O-rings that are part ofthe gasket but raised sufficiently to form a seal onto the cartridgewhen pressed against it with the clamp part. The gasket is illustratedin FIG. 8.

5) Biochip

The fabrication of chips having holes for ion transport measurement isdescribed elsewhere. In this device, the chip is made of glass and has16 laser drilled holes. The chip is laser polished on the top surface,and treated in acid and base prior to attaching the chip to an upperchamber piece with a UV adhesive.

6) Clamp Assembly:

A clamp is made from an inflexible material so as to not allow bowing ofthe cartridge during compression onto the gasket while mounted on thetester. In this case it is made of stainless steel for its inertnesswhen wetted with physiological buffers. The clamp is shaped so as to fitsnugly over the cartridge and is drilled so as to accommodate and bepositioned by the guide-pins sticking out of the inner chamber array.Four screws are finger-tightened to the base plate at each corner of theclamp assembly so as to press down the cartridge to seal it against thegasket. This part is shown in FIG. 10.

7) Upper Chamber Electrodes:

In early development it was expected that compression pins would contactthe bottom of the cartridge during testing to provide a connection tothe reference electrodes built in to the cartridge. The presentembodiment of the cartridge does not contain reference electrodes,therefore these electrodes must be introduced into the top wells of thecartridge. To this end, periodically chlorided silver wires are used aselectrodes. The electrodes are shaped to dip deep inside each well, andon the outside of the wells are soldered to a wire running along the topof the clamp part. At each end of this wire is a 1 mm female pinconnector that is used to interface with the voltage clamp amplifier.The electrode array is pictured in FIG. 5.

Method:

Before use the device should be clean and dry.

A SealChip cartridge is removed from its carrier, and rinsed with a jetof deionized water of approximately 18 MOhms resistance. The product isthem dried under a stream of pressurized dry air filtered through a 0.2μm air filter to remove water from the recording apertures and theirvicinity.

The clean cartridge is then placed with top-wells upward onto thepressure contact pins of the tester such that movement of the cartridgeis limited by the six alignment pins of the bottom-chamber. Thecartridge should be supported above the PDMS gasket but without yettouching the gasket. The clamp is them placed over the cartridge suchthat the 4 mounting holes align with their threaded counterparts on thebase plate. The 4 mounting screws are them used to press down the clampuniformly thereby pressing the cartridge down onto the PDMS gasket withsufficient pressure to form a tight seal between the chip and the gasketand between the gasket and the bottom chamber array. The recordingaperture within each chamber of the cartridge should already be alignedwith openings in the gasket that form the bottom chambers.

The bottom chambers are then filled from one side with sufficientsolution (analogous to intracellular solution) to fill the bottomchamber and fill enough of the tubing on the other side such thatcapacitative distension of the tubing on the filling side will notintroduce air into the recording chamber, and will not introduce airinto the area of the tubing that contains the bottom-chamber electrode.For this purpose, it is best to fill the chamber starting from the sidethat does not contain the electrode since higher pressures will be usedfor vacuum pressure than for positive pressure, thereby ensuring thatthe electrode will remain in full contact with the solution at alltimes. Once the bottom chamber is filled and is free from visiblebubbles, the tubing should be sealed off by a clamp or valve or anymeans that ensures electrical isolation between the bottom chambers ofthe array. Sufficient positive pressure should be applied to the freeend of the inner chamber tubing so as to cause solution to be forcedinto the counterbore and through the hole of the recording aperture ofthe chip.

Once solution is seen emerging into the top chamber, the pressure shouldbe released, and immediately the top chamber should be filled withsufficient solution (analogous to extracellular solution) so as tocompletely immerse the top side of the chip without bubbles remaining onthe chip surface, and fill the top well sufficiently to provide goodcontact with the electrode in the top well. It is also of benefit tofill the top well sufficiently to avoid a strong meniscus effect (60 to70 μL with the present version of the SealChip product) whenever it isintended to view under an inverted microscope the progression of theexperiment (for upright microscopes it is necessary to fill with moresolution, ˜90 μL, to allow good contact with a coverslip that must beplaced over the well to enable a good view of the bottom of the well).

The assembled tester is now ready for testing and should be placed onthe microscope (or appropriate shielded location if observation of theexperiment is not necessary) and connected to the voltage clampamplifier(s) as well as to the pressure control device(s) for testing.

After the termination of the experiment, the tester is disconnected andremoved from its testing location. The extracellular medium is suctionedfrom each well, and each well is rinsed once with deionized water toremoved any leftover particulate (debris or cellular) material that maybe left over from the experiment. Both ends of the tubing of the bottomchambers are then opened and the solution is suctioned out of the bottomwells. Each well should be well rinsed with clean deionized water, thendried completely with pressurized air. Finally the screws holding downthe clamp are removed and the cartridge is disassembled from the tester.Any wetting at the gaskets should be wicked away with a lint-freetissue. If any liquid is pooled around the gasket, then the gasketshould be removed, rinsed then dried, and the bottom chamber arrayshould be likewise rinsed and dried, ensuring that the tubing is alsorinsed and completely dried.

Quality Control/Quality Assurance of SealChip Product:

Internally to the company, the tester has been used for QC/QA of theSealChip product before it is sent to customer, and before it is usedinternally for further research. The success rate with product thatpasses the QC has been as good as that with older testers that tested asingle chamber at a time.

Quality Control/Quality Assurance of Cells:

Internally to the company, the tester has been used to verify thequality of the cells used for QC/QA using known good SealChip product.

Research and Development:

The tester has been used by our company for testing variations to theSOP for the SealChip product. In the future it may be used for discoveryand screening of compounds that require exchanging of solutions on thebottom well or where compounds or particles must be delivered to thecytosolic chamber after a seal is formed with the cell membrane.

A great number of results have been achieved on the microscope adapteddevice (“Tester Unit”) since its discovery. The tester was verified ascomparable to the original tester based on a single-aperture test inDecember, 2002. Since January 14^(th) it has been the tool of choice forperforming quality control experiments on the SealChip product. Thefollowing is examples of the quality of data obtained from it. *SealChipData Since Jan 14th, 2003 Cell Chip Lot# Hole ID Type Re(G) Rm(G) Ra(M)Seal Qty Note S2N22-40 C RBL 3.4 0.5 5.7 +++ G 3.3 5 6.7 +++ I 3.3 2 2+++ M 3.2 0.25 8.8 +++ O 3.2 0.5 6.5 +++ S2D18-114 A 3.9 2.4 7.2 ++ C3.9 2.2 18 + G 3.7 4 10.8 ++ S2D20-28 B 4.5 2.6 9.1 ++ C 4.2 1 13.3 −S D4.4 0.6 10.5 + E 4.3 2.7 10 ++ F 4.3 1.6 10 ++ G 4.2 3.5 9.4 ++ H 4.33.3 8.8 ++ S2D20-8 A 4.1 1.7 12.2 ++ C 4.1 2.7 9.3 ++ G 4.2 1.7 8.4 ++ I4.1 2 11 + M 4.1 1.6 11.7 S Debris landed before cell O 4.1 2.6 7.6 ++S2D20-50 A 4.3 2.9 12.4 + B 4.3 7 10.7 +++ C 4.1 1.1 10 +++ D 4.3 2.18.8 +++ E 4.2 4.5 8 ++ F 4.4 4.9 7.1 ++ G 4.3 1.5 10 ++ H 4.3 6.9 8.3 ++I 4.2 6.2 8.3 +++ J 4.2 0.6 8.1 +++ K 4.3 0.9 9.8 ++ L 4.4 6.5 7.4 +++ N4 6 7.7 +++ O 4 5.6 7.8 ++ P 4.1 6.5 12.8 +++ S2D219-21 D 3.1 4.5 4.6+++ E 3 1.5 11.6 + F 3 1.5 5.6 ++ G 3 2.8 5.8 +++ H 3 3.1 4.8 +++ I 33.2 8 ++ J 3.1 3 5.7 ++ S2D18-191 A 3.5 3.3 8.5 ++ C 3.5 2 13.9 ++ D 3.31.6 8.9 ++ E 3.6 2.5 9.2 +++ F 3.6 2 8 +++ G 3.5 0.4 7.7 +++ H 3.7 1.4 6++ S2D18-206 A 3.3 4.1 7 +++ C 3.2 2.1 6.2 ++ D 3.3 4.6 6.7 ++ E 3.4 3.45.2 ++ F 3.1 0.7 5.8 +++ H 3.4 0.6 11 S S2D20-6 B 4.1 1.5 8.8 ++ C 4.30.5 8.9 ++ D 4.1 3.2 8.9 +++ E 4.1 3.3 6.8 +++ G 4.3 3.8 7.8 +++ H 4.33.2 10.4 +++ S2D20-133 A 4.3 3.5 9.6 S B 4.5 4.4 7.5 ++ C 4.4 5 11.4 ++D 4.5 3.1 10.8 + E 4.5 5.3 10 +++ F 4.4 5.1 8.8 ++ G 4.4 5.1 8.5 ++ H4.3 1.1 10.5 + S2D21-70 A 4.2 2.1 22 + Spec near the hole B 4.2 2.7 8+++ C 4.3 2.8 7.6 +++ D 4.3 1.3 12.3 ++ E 4 2.3 10.2 ++ F 4.2 0.5 7.2+++ S2D20-130 A 3.2 0.8 7.6 + B 3 0.5 8.9 ++ E 3 1.3 11.1 ++ F 3.3 2 7.9+++ G 3.3 0.5 11.9 S H 3.1 2.1 7.8 ++ S2D20-194 A 3.7 2.3 9.8 +++ C 3.63 7.9 ++ D 3.8 2.4 14 S E 3.6 2.4 5.9 ++ F 3.9 2.1 12.1 ++ G 3.7 2.1 6.7+++ H 3.8 0.9 8.3 ++ S2D18-81 A 3 1.6 5.5 ++ C 3.1 2.1 6 ++ D 3.3 3.47.8 ++ E 3.3 2 6.6 ++ F 3.3 2.4 8.6 ++ G 3.4 2.9 8.6 + H 3.3 2.8 5.7 +++S2D20-171 C 3.8 2.3 9.5 ++ E 3.8 2.7 8.3 ++ F 3.9 3.4 8.1 ++ G 3.7 3.36.2 +++ H 3.7 2.8 7.8 +++ I 3.7 2.8 12.7 + J 3.8 3.3 5.9 +++ S2D16-26 A3.3 1.5 5.5 ++ C 3.5 1.9 7.5 +++ D 3.7 1.2 6.8 ++ E 3.5 1.7 7.5 +++ F3.7 1.7 6.4 +++ H 3.7 1.7 8.8 ++ S2D19-20 A 2.5 1.4 5.7 ++ C 2.5 1.8 4.5+++ D 2.5 1.5 5.8 ++ E 2.5 1.1 5 ++ F 2.4 1.8 1.6 +++ G 2.7 1.4 4.8 ++ H2.8 1.6 5 ++ S2D16-1 B 3.2 1.2 10.3 S C 3.1 1.6 6.5 + D 3.1 0.6 17 S E2.9 2.3 6.1 ++ F 3.1 2.7 6.1 ++ G 3.1 2.7 7.8 +++ S3210-181 A Cho-Herg4.6 0.3 14 +++ B 4 0.5 11 +++ D 4 0.2 14 ++ E 4 1.3 17 ++ G 4 2.1 10 +++H 4.1 0.6 12 S S3214-60 A 3.6 1.2 7 ++ B 2.9 1 7 +++ C 2.9 0.4 17 −S D2.9 1.3 11 + G 3.1 1.7 10 +++ H 3 0.2 10 ++ 031103-A1 B RBL 3 1 4 ++ D3.4 0.5 5.2 ++ F 3.1 1.1 4.1 +++ H 3 1.2 7 ++ N 3.2 0.4 4.4 ++ P 3.1 0.35.5 + 031103-A2 A 3.8 0.6 4.1 ++ C 4.3 2.1 4.1 ++ D 4 2.3 8.1 ++ K 4.42.1 5.3 +++ M 4.8 2.3 7.8 ++ O 4.4 2.7 9.9 ++ 030703-A1 A 3.6 1.9 4.9 ++C 3.7 2.3 3.6 +++ E 3.8 2.2 5.8 ++ G 3.7 1.8 5.2 ++ I 3.4 1.7 4.1 ++ M3.5 2.1 5.4 ++ O 3.7 1.7 4.6 ++ 031103-A3 A 4.8 2.5 5.2 ++ C 4.6 1.4 5.4++ E 4.8 1 4.6 ++ I 4.9 0.3 6.1 ++ D 4.9 1.6 6.1 ++ F 4.9 0.7 8.1 ++030603-A2 B 4.3 1.2 4.2 +++ C 4.3 4 9.2 ++ F 4.3 2 8.2 ++ H 4.4 2.2 7 ++G 4.6 2 7.7 +++ I 4.2 2.2 7.2 +++ J 4.3 1.2 5.8 ++ 030603-A1 A 4.4 1.86.4 + B 4.4 1.2 8 ++ C 4.6 1.2 8 + F 4.8 1.5 8.5 + G 4.4 0.7 4.4 ++ H4.3 1.4 5.9 + I 4.2 1.1 8.6 ++ 030603-A3 B 4 1.7 6.9 +++ D 4 0.28 6.9 ++F 4.2 0.35 4.4 ++ H 4.3 0.27 6.9 ++ L 4.4 0.25 7.2 ++ N 4.5 0.85 7.2 ++

Example 6 Cell Preparation for Ion Transport Measurement

PART I. CHO wt. and CHO.Kv cells 1. Use cells @ 50%˜70% confluency. (18hrs after cells seeded 1:10˜1:15) 2. Remove medium and wash x2 withX⁺⁺-free PBS (extra wash might be necessary if the final cell suspensionhas too much small debris) 3. Treat for 2′15″ with 1:10 trypsin-EDTA, atthis time the supernatant might be a little turbid due to release ofcells into the buffer. 4. Rock gently, aspirate to discard supernatant.Wait for 1′25″. 5. Add 1 volume of X⁺⁺-free DMEM complete with 10% FCS,NEAA, etc, rock gently to loosen and detach cells, and spin down (do nottry to blow to remove the remaining cells sticking to the bottom) 6.Wash x1 with PBS complete 7. Resuspend in PBS, triturate, and passthrough 15˜20 μm filter into non-stick plate. Cells can be used after 10minutes of recovery and should last for up to 4 hr PART II. Transientlytransfected CHO cells. 1. Remove medium and wash x2 with X⁺⁺-free PBS 2.Treat for 1′ with 5 ml 1:10 trypsin-EDTA (0.5 ml 0.05% trysin 0.53 mMEDTA from GIBCO cat. No. 25300-54 in 4.5 ml PBS) 3. Rock gently,aspirate to discard supernatant. 4. Add 0.5 ml fresh 1:1 trypsin-EDTA,Wait for 6 mins. 5. Add 5 ml of X⁺⁺-free DMEM complete with 10% FCS,NEAA, etc, rock gently to loosen and detach cells, leave cell at RT for1 hour, and spin down (do not try to blow to remove the remaining cellssticking to the bottom) 6. Wash x2 with 1 ml PBS complete 7. Resuspendin PBS, triturate, and pass through 15 to 20 micron filter intonon-stick plate. Part III. CHO-Herg cells. 1. Use cells at 50%˜70%confluency in T-25 flasks (VWR, Cat. No. 29185-302). 2. Remove mediumand wash x2 with X⁺⁺-free PBS (extra wash might be necessary if thefinal cell suspension has too much small debris) 3. Treat for 1′ with 2ml trypsin-EDTA(0.5 ml 0.05% trysin 0.53 mM EDTA from GIBCO cat. No.25300-54 in 1.5 ml PBS) 4. Rock gently, aspirate to discard supernatant.Wait for 2 mins. 5. Add 5 ml volume of X⁺⁺-free DMEM complete with 10%FCS, NEAA, etc, rock gently to loosen and detach cells, leave cell at RTfor 30 min, and spin down (do not try to blow to remove the remainingcells sticking to the bottom) 6. Wash x2 with 1 ml PBS complete 7.Resuspend in PBS, triturate, and pass through 15˜20 μm polyester filterinto non-stick plate if cells still cluster together.

Part IV. Protocol for Isolation of CHO

1. Use cells at 70˜80% confluences in T-25 flasks (24 hrs afterseeding).

2. Remove medium and wash×2 with X⁺⁺-free PBS ((cell should not be leavein X⁺⁺-free PBS more than 10 mins, otherwise, the minimal digestion timewill be decreased)

3. Wash once with 1:4 AccuMax (wait about 20 second, rocking to removedthe loose attached cell)

4. Treat at 37° C. w 4 ml volume of 1:4 Accumax (diluted with X⁺⁺-freePBS) for minimal time (cell dissociate from the flask and floated in theAccumax) or 1.5 times minimal time.

CHO-KV

a. 1:4 AccuMax 5′ (1 ml AccuMax+3 ml X⁺⁺-free PBS) w/o rocking

b. 1:4 AccuMax 8′ (1 ml AccuMax+3 ml X⁺⁺-free PBS) w/o rocking

CHO-HERG

c. 1:4 AccuMax 8′ (1 ml AccuMax+3 ml X⁺⁺-free PBS) w/o rocking

d. 1:4 AccuMax 12′ (1 ml AccuMax+3 ml X⁺⁺-free PBS) w/o rocking

5. Add 5 ml volume of Ca⁺⁺-free DMEM with 10% FBS, into the flasks, andremoved all cell suspension to a 15 ml centrifuge tube, spin down ˜300g×3 min (do not try to blow to remove the remaining cells sticking tothe bottom).

6. Discard supernatant, add 1 ml 1:4 (PBSC:PBS), gently triturate toresuspend cell, centrifuge 2000 rpm×1 min in an micro centrifuge tube.

7. Discard the supernatant, add 800 μl to 1 ml 1:4 (PBSC*:PBS),triturate, and pass through 15˜20 □m filter into non-stick plate. DMEMw/o Gibco 500 ml 21068-  4° C. Ca++ Innovative 028 cell AccuMax Tech 100ml AM105 $36.00 −20° C. Dubbecco's Gibco 500 ml 14287- RT 1X PBS (PBSC*)080 Dubbecco's VWR 500 ml 16777- $9.50 RT 1X PBS X⁺⁺-free 149 FBS Gibco500 ml 10082- inquire −20° C. 147 24 well non- VWR 29443-032 176.55stick plate

Part V. Protocol for Isolation of HEK

1. Use HEK-Na cells at 7080% confluences in T-75 flasks (16 hrs afterseeding).

2. Remove medium and wash×2 with X⁺⁺-free PBS

3. Add 6 ml X⁺⁺-free PBS, incubate at 37° C. for 5 mins, aspiratesupernatant

4. Add 6 ml X⁺⁺-free PBS, incubate at 37° C. for 10 mins or until allcells dissociate from flask.

5. Add 2 ml Accumax directly into flask to finalize the Accumaxconcentration to 1:4, incubate cell at 37° C. for 4 mins

6. Add 6 ml volume of Ca⁺⁺-free DMEM with 10% FCS into the flasks tostop the digestion

7. Put cell mixture into a 15 ml tube, and spin down 300 g×3 min

8. Discard supernatant, gently suspend cell in 4 ml Ca⁺⁺ free DMEM with10% FCS, incubate cell at 37° C. incubator at least 30 mins or until useit.

9. Carefully remove the supernatant, wash×1 with PBS with 100 nM Cacl₂,1 mM Mgcl₂

10. Triturate, resuspend cell in PBS with 100 nM Cacl₂, 1 mM Mgcl₂,filter cell mixture through 21 μm filter into non-stick plate.

Example 7 Program Logic and Pressure Profile

The following is a typical program logic for software pneumatic control.It includes procedures for cell landing, form seal, break-in, and Racontrol. #start of program Count=0 Turn off compensations ProcedureLanding:  Reset button_pressed  Label window “Attempting Landing”  Runwasher # deliver clean ES to top chamber  Wait 5 seconds  Stop washer Repeat twice:   Apply −300torr pressure # clear holes of any remaining  debris after filling   Wait 0.5 seconds   Apply 0torr pressure   Wait2 seconds  End repeat  Zero junction potential  Wait for stable reading Record average Re value  Save Re to logs  Initiate cell addition  Waituntil 0.5 seconds before cell delivery # before pipette touches ES Apply +10torr # before and during delivery  Wait for pipette removal #from ES chamber  Apply 0 torr  Wait 3 seconds  Apply −50torr  Wait untilSeal > 2Re for 0.5sec or elapsed=15 seconds  If elapsed then  Count=count+1   If count >= 3 then abort test and write to log   Apply+50torr   Run proc Landing  Endif  Run FormSeal End procedure Resetelapsed Procedure FormSeal  Reset button_pressed  Label window“Attempting Seal”  Apply −80mV HP #negative holding immediately afterlanding  Apply −50torr #this may not necessarily be the same as that used for landing  While Seal increasing >20MOhms/second   Wait untilSeal >= 1Gohm or elapsed=10 seconds  Endwhile  Apply 0torr  Wait 2seconds  While seal increasing >20MOhms/second and seal<1GOhm,   Wait 1second  Endwhile  #start ramping to attempt seal  Unless seal>1GOhm,Apply ramp from 0torr to −50torr over 20 seconds  Unless seal>1GOhm,Wait 5 seconds  Unless seal>1GOhm, Apply 0torr  Unless seal>1GOhm, wait5 seconds  Unless seal>1GOhm, Apply ramp from −30torr to −80torr over 30 seconds  Unless seal>1GOhm, Wait 5 seconds  Unless seal>1GOhm, Apply0torr  Unless seal>1GOhm, wait 5 seconds  Unless seal>1GOhm, Apply rampfrom −50torr to −100torr over 40  seconds  Unless seal>1GOhm, Wait 5seconds  Unless seal>1GOhm, Apply 0torr  Unless seal>1GOhm, wait 5seconds  Unless seal>1GOhm, Apply ramp from 0torr to −200torr over 120 seconds  Unless seal>1GOhm, Wait 5 seconds  Unless seal>1GOhm, Apply0torr  Unless seal>1GOhm, wait 5 seconds  If not seal>1GOhm    Checkbutton_pressed    If button_pressed = “continue” then abort test andwrite to log    Run FormSeal  Endif  #Seal detected, now check stability Stop ramping and hold last pressure  Wait 1 second # let seal stabilize If seal>1GOhm,   Apply 0torr   Record Seal value into Rseal, save tologs   Unless Seal<(Rseal−200MOhms) or Seal decreasing  >200MOhms/second    Wait 5 seconds   End unless   IfSeal<(Rseal−200MOhms) or Seal decreasing >200MOhms/second    Checkbutton_pressed    If button_pressed = “continue”, goto Procedure BreakIn   Run FormSeal   Endif   #cell sealed  Endif End Procedure ProcedureBreakIn:  Reset button_pressed  Label window “Attempting break-in”  Nullchamber capacitance  Until capacitance > 3.5pF or Pressure>300torr orSeal<(Rseal−200MOhms) or Seal decreasing >200MOhms/second   Wait 1second   Apply −20 delta torr  End until  If capacitance > 3.5pF  Record break-in pressure value   Wait 0.5 seconds   Apply 0torr   Runprocedure RaControl  Endif  If Pressure>300torr   Apply 0torr   Untilcapacitance > 3.5pF or Pressure>300torr or  Seal<(Rseal−200MOhms) orSeal decreasing >200MOhms/second    Wait 1 second    Apply −20 deltatorr    Apply Zap   End until   If pressure>300torr then abort test andwrite to log  Endif  If capacitance > 3.5pF   Record break-in pressurevalue   Wait 0.5 seconds   Apply 0torr   Run procedure RaControl  Endif If Seal<(Rseal−200MOhms) or Seal decreasing >200MOhms/second   Checkbutton_pressed   If button_pressed = “continue”, goto Procedure BreakIn  Run FormSeal  Endif End Procedure Elapsed = 0 Procedure RaControl: Reset button_pressed  Label window “Adjusting seal quality”  Record Cm,Rm, Ra to logs  Assign RmInitial = Rm, RaInitial = Ra  If Ra < RaIdealthen end #RaIdeal does not need adjustment  If Ra < RaMax and Radecreasing then end #no need for adjustment  If Ra < RaMax thencountdown = 20 seconds else countdown = “true”  While countdown   Checkbutton_pressed   If button_pressed = “continue” then end   If Raincreasing and Rm > 300MOhms    Apply −50torr    Wait 0.5seconds # max 2seconds    Apply 0torr    Wait 1.5 seconds   Endif   If Ra increasingand Rm > 500MOhms    Apply −80torr    Wait 0.5seconds # max 2 seconds   Apply 0torr    Wait 1.5 seconds   Endif   If Rm>0.8GOhm then apply−50torr else apply −10torr   While Ra>RaIdeal and Rm>(RmInitial−25%) andcountdown    Unless Ra<RaIdeal or Rm<(RmInitial−25%), wait 5 seconds   If Ra<RaMax then countdown=20 seconds    If Ra<RaIdeal then Endwhile   If Ra not decreasing     If Rm not decreasing and Rm>1GOhm then Apply−10 delta torr     If Rm not decreasing and Rm<1GOhm then Apply −5 deltatorr     If Rm decreasing and Pressure>10torr then Apply +5 delta torr    If Rm<(RmInitial−25%) then apply 0torr    Endif    Ifpressure>BreakInPressure then apply 0torr    If elapsed > 120 secondsthen apply 0torr and end    If Rm<300MOhms then apply(reakInPressure−10torr)   Endwhile   If −10torr>pressure>−50torr   Apply 0torr    If Ra increasing then apply −60torr    If Raincreasing then run RaControl Procedure   Endif  Endwhile End Procedure

Example 8

The pressure profile of Example 6 was employed in a 52-cell test asdescribed in Example 2. The criteria for the test was the achievement ofat least 75% success rate, defined as achieving a gigaohm seal toinitiate a patch clamp, then during the patch clamp membrane maintainingresistance above 200 MOhms and maintaining access resistance (or seriesresistance) below 15 MOhms for at least 15 minutes. Table 1 demonstratesthe conclusion from this experiment, showing that the goals of the52-cell test were met. FIG. 27 gives a sample of the time-course of anexperiment where membrane resistance and access resistance values arekept within the required parameters. At many locations in the recordingthere are deflections in the access resistance trace. These deflectionsrepresent locations where the pressure protocol was applied to maintainthe seal quality parameters. The success rate at achieving gigaohm sealsis demonstrated in FIG. 24. This data is a graphical representation ofthe data identified in Table 1, where 90% of the chips produced agigaohm seal with CHO cells. FIG. 25 shows a histogram of the parametersachievable with this pressure control protocol. Data shown in bluerepresents initial values for Ra and Rm, and values in red representvalues for Ra and Rm after 15 minutes of continuous whole-cell accessunder voltage clamp conditions. These data demonstrated that overall,75% of the cells achieved gigaohm seals, and then whole-cell access wasattained with acceptable parameters that were well-controlled for atleast 15 minutes.

Example 9 Single Channel Recording using a Biochip Comprising a Hole forIon Transport Measurement

RBL cells were prepared for patch clamp recording by simplecentrifugation. The cells were then delivered onto an ion transportmeasurement device with a single recording aperture. The biochip devicewas assembled according to Example 2. The biochip had been treated withacid and base to improve sealability. The upper chamber solution was PBSlacking calcium and magnesium. The lower chamber solution was: 150 mMKCl, 10 mM HEPES-K, 1 mM EGTA-Na, 1 mM ATP-Mg pH (KOH) 7.4 for A throughC in FIG. 22, the lower chamber solution was:

8 mM NaCl, 20 mM KCl, 1 mM MgCl₂, 10 mM HEPES-Na, 125 mM K-Glu, 10 mMEGTA-K, 1 mM ATP-Mg pH (KOH) 7.2 for D in FIG. 22.

Seal formation was achieved as provided in the previous examples, butafter gigaseal formation, no break-in step was performed. Single-channelrecordings were obtained from a cell-attached membrane patch on an RBLcell. An inward rectifier IRK1 single channel was recorded in RBL cells.A low concentration of extracellular K⁺ which does not depolarize thecell and does not inactivate the channel was used. ATP was present inthe internal solution, which prevents the rundown of the channelactivity. The noise level of the recordings was reduced from 10 pA to 1pA in order to observe single channel events, which have an amplitude ofa few picoamps.

1. An ion channel chip comprising: a. a planar polymer plastic substratetreated with an ionized gas; b. at least one aperture positionedsubstantially perpendicular to said planar polymer plastic substrate; c.a gasket positioned about said aperture defining the perimeter of achamber; and wherein said treatment forms at least one functional groupor moiety positioned sufficiently close to said aperture such that acell can simultaneously contact said at least one functional group ormoiety and said aperture.
 2. The ion channel chip according to claim 1,wherein said polymer plastic is selected from the group consisting of apolyimide, a polyolefin and a polyacrylate.
 3. The ion channel chipaccording to claim 1, wherein said polymer plastic is comprises amaterial selected from the group consisting of silicon, a wax, aparaffin wax, Parafilm(™), and a fatty acid polymer.
 4. The ion channelchip according to claim 1, wherein said substrate comprises a thicknessbetween 5 mM and 300 mM.
 5. The ion channel chip according to claim 1,wherein said ionized gas is a plasma gas.
 6. The ion channel chipaccording to claim 1, wherein said at least one aperture has a diameterless than or equal to a diameter of a cell.
 7. The ion channel chipaccording to claim 1, wherein said at least one aperture comprises atleast 96 apertures or at least 1536 apertures.
 8. The ion channel chipaccording to claim 1, wherein said functional group provides a negativecharge or a hydrophilic property.
 9. The ion channel chip according toclaim 8, wherein said functional group comprises moiety selected fromthe group consisting of a sulfate, a phosphate, a borate, a silicate, acarboxyl, a thiosulfate, a thiophosphate and a thiocarbonate.
 10. An ionchannel chip comprising: a. a planar substrate; b. at least one aperturepositioned substantially perpendicular to said planar substrate; c. alipid positioned about said aperture, wherein a cell may continuouslycontact a portion of said lipid and said aperture.
 11. The ion channelchip according to claim 10, wherein said substrate comprises polymerplastic or glass.
 12. The ion channel chip according to claim 10,wherein said lipid comprises a tail portion bound to said chip and ahead portion capable of interacting with a cell.
 13. The ion channelchip according to claim 10, wherein said lipid is a lipid bilayercomprising a first lipid layer and a second lipid layer, each lipidlayer comprising a tail portion and a head portion, further wherein saidfirst head portion is attached to a surface of said chip; furtherwherein said first tail portion interacts with said second tail portionby van der Waals forces; and further wherein said second head portion iscapable of interacting with a cell.
 14. The ion channel chip accordingto claim 13, wherein said lipid bilayer comprisesphosphatidylethanolamine lipid.
 15. The ion channel chip according toclaim 10, wherein said lipid is in contact with the inner surface ofsaid aperture.
 16. A method of manufacturing a preassembled iontransport measurement cartridge comprising: a. providing a cartridge anda chip substrate; b. attaching said chip substrate to said cartridge; c.drilling a throughbore through said chip substrate; d. modifying thesurface of said chip substrate; wherein said modification increased theability of said chip substrate to form a tight seal with a cell.
 16. Themethod according to claim 15, wherein said modifying the surfacecomprises exposing said chip substrate to an acid or a base.
 17. Themethod according to claim 15, wherein said modifying the surfacecomprises exposing said surface to an ionized gas.
 18. An ion channelchip comprising: a. a planar polymer plastic substrate; b. a glasssurface layer positioned on opposing sides of said planar polymerplastic substrate; and c. at least one aperture positioned perpendicularto said glass surface layers and said polymer plastic substrate.
 19. Atransportation system for an ion channel chip comprising: a. a fluidtight container capable of accepting an ion; b. an ion channel chipaccording to claim 1; and c. a fluid.
 20. A method of laser drilling anion channel chip comprising: a. providing a chip substrate; whereinregions of said chip substrate not to be laser drilled are masked b.providing a laser; c. splitting the beam from said laser into two ormore beamlets; and d. focusing said beamlets through at least one lensonto said chip substrate thereby creating a throughbore or counterbore.21 The method according to claim 20, wherein said masked regions areregions covered with a material selected from the group consisting ofchrome coated quartz, aluminium coated quarz, dielectric coated quartzand metal.
 22. The method according to claim 20, further comprisingpretreating said chip substrate by exposing said chip substrate to a UVlaser.
 23. A method of manufacturing a substrate for an ion channel chipcomprising: a. treating a first glass surface with an acid; b. treatinga second glass surface with a base; c. contacting said first glasssurface with said second glass surface; d. heating said surfaces to atemperature capable of bonding said first glass surface to said secondglass surface.
 24. A method of manufacturing a substrate for an ionchannel chip comprising: a. treating a first glass surface with a base;b. treating a second glass surface with a base; c. applying Na2SiO3 tosaid first glass surface; d. contacting said second glass surface tosaid applied Na2SiO3; and e. heading said surfaces to a temperaturecapable of bonding said Na2SiO3 to said glass surfaces.
 25. A vacuumgasket for use with an ion channel chip comprising: a. a planar surfacehaving a length and width approximately equal to the length and width ofat least one ion channel chip; b. a sealing structure protrudingparallel to said planar surface, wherein said sealing structure iscapable of encircling the perimeter of an aperture of said ion channelchip when engaged; c. a vacuum groove permitting the removal of air frombetween said planar surface and said chip thereby creating a vacuumseal; and wherein said sealing structure defines a chamber when saidgasket is sealed to said ion channel chip.
 26. An automated system forprocessing of ion channel chips comprising: a. a chip manipulatingstructure capable of engaging and manipulating the ion channel chipaccording to claim 1, b. a negative pressure structure capable ofproviding negative pressure to said ion channel chip; c. a positivepressure structure capable of providing positive pressure to said ionchannel chip; and d. a storage structure for storing said ion channelchip.
 27. A storage structure for an ion channel chip comprising: a. atop structure; b. a bottom structure comprising a series of chipengaging structures, wherein each engaging structure is capable ofengaging an ion channel chip; further wherein said bottom structurereversible engages said top structure.